Specific Effects of Dietary Methylmercury and Inorganic Mercury in Zebrafish (Danio rerio) Determined by Genetic, Histological, and Metallothionein Responses

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Specific Effects of Dietary Methylmercury and Inorganic Mercury inZebrafish (Danio rerio) Determined by Genetic, Histological, andMetallothionein ResponsesSophie Gentes,† Regine Maury-Brachet,† Caiyan Feng,∥ Zoyne Pedrero,∥ Emmanuel Tessier,∥

Alexia Legeay,† Nathalie Mesmer-Dudons,† Magalie Baudrimont,† Laurence Maurice,⊥,§

David Amouroux,∥ and Patrice Gonzalez*,‡

†Universite de Bordeaux, EPOC, UMR CNRS 5805, Place du Dr B. Peyneau, F-33120 Arcachon, France‡CNRS, EPOC, UMR 5805, F-33120 Arcachon, France∥Laboratoire de Chimie Analytique, Bio-Inorganique et Environnement, Institut des Sciences Analytiques et de Physico-Chimie pourl’Environnement et les Materiaux (IPREM), CNRS-UPPA-UMR-5254, Helioparc, 2 Avenue du President Pierre Angot, F-64053 Pau,France§Observatoire Midi-Pyrenees, Laboratoire de Geosciences Environnement Toulouse, Universite Paul Sabatier Toulouse III, 14 avenueEdouard Belin, 31400 Toulouse, France⊥GET, IRD, F-31400 Toulouse, France

*S Supporting Information

ABSTRACT: A multidisciplinary approach is proposed hereto compare toxicity mechanisms of methylmercury (MeHg)and inorganic mercury (iHg) in muscle, liver, and brain fromzebrafish (Danio rerio). Animals were dietary exposed to (1) 50ng Hg g−1, 80% as MeHg; (2) diet enriched in MeHg 10000 ngHg g−1, 95% as MeHg; (3) diet enriched in iHg 10000 ng Hgg−1, 99% as iHg, for two months. Hg species specificbioaccumulation pathways were highlighted, with a preferentialbioaccumulation of MeHg in brain and iHg in liver. In thesame way, differences in genetic pattern were observed for bothHg species, (an early genetic response (7 days) for both speciesin the three organs and a late genetic response (62 days) foriHg) and revealed a dissimilar metabolization of both Hgspecies. Among the 18 studied genes involved in key metabolic pathways of the cell, major genetic responses were observed inmuscle. Electron microscopy revealed damage mainly because of MeHg in muscle and also in liver tissue. In brain, high MeHgand iHg concentrations induced metallothionein production. Finally, the importance of the fish origin in ecotoxicological studies,here the seventh descent of a zebrafish line, is discussed.

■ INTRODUCTIONMercury (Hg) is a toxic trace metal naturally present indifferent terrestrial compartments but human activities areclearly the major source of its remobilization in the environ-ment.1,2 Exposure and uptake of Hg in aquatic organismsoccurs mainly through food.3,4 Therefore, the ichthyofaunarepresents a significant source of Hg for humans because of thecontamination of aquatic ecosystems.5

Chemical speciation of Hg defines its absorption, assim-ilation, distribution, and toxicity within an organism.6

Methylmercury (MeHg) is the most toxic species of Hg dueto its ability to cross biological membranes: it forms a complexwith cysteine and enters the cell using neutral amino acidcarriers.7 Another characteristic of MeHg is its capacity forbiomagnification along foodwebs.8,9 In humans, the absorptionefficiency of MeHg is 95% regardless of the route of exposure

and about 10% for inorganic Hg (iHg). While crossingbiological barriers with difficulty, iHg still remains toxic, butat higher concentrations. Absorbed MeHg is assimilatedthrough the gastrointestinal barrier, then passes into theblood where it is distributed into target organs, mainly brainand muscle.7 Chronic consumption of MeHg-contaminated fishcan cause adverse health effects in humans, especially on thecentral nervous and immune systems.10,11 In contrast, iHgtends to be accumulated in detoxification organs (liver andkidney).12 Only a few studies have looked at iHg toxicity,although many studies have been done on MeHg.13 However,

Received: July 24, 2015Revised: October 19, 2015Accepted: October 28, 2015Published: October 28, 2015

Article

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© 2015 American Chemical Society 14560 DOI: 10.1021/acs.est.5b03586Environ. Sci. Technol. 2015, 49, 14560−14569

regulatory mechanisms, transport, metabolism and eliminationof Hg species in higher organisms such as mammals, includinghumans, are still far from being fully explained.6,7,14 Eventhough in recent years, the structural identity of somebiomolecules containing Hg has been elucidated,15−19 provid-ing valuable information about its potential role in Hgdetoxification, there is still a lack of knowledge of Hg pathwaysin living organisms. In cells, MeHg generates oxidative stressthrough reactive oxygen species (ROS) formation. It interactswith cysteine sulfhydryl groups, disrupts calcium ion homeo-stasis, causes damage such as mitochondrial disturbance, lipidperoxidation, DNA damage, membrane structure alteration, cellcycle dysfunction and apoptosis, damage to the immune systemand glucose metabolism disturbance.6 Several studies haveobserved these changes through transcriptome study of adultzebrafish (Danio rerio) and fathead minnow (Pimephalespromelas) (two biological models) contaminated with differentlevels of MeHg (acute, sublethal, and chronic) and via thedirect or the trophic pathways in muscle, brain, and liver.20−24

Cells also have antioxidant enzymes to fight against oxidativestress, like superoxide dismutase and glutathione peroxidase.25

Other defense mechanisms exist such as active efflux pumpsbelonging to the family of ATP binding cassette (ABC)transporters26 or metal sequestration proteins like metal-lothioneins. These low molecular weight cysteine-rich proteinsare able to chelate and scavenge metals.27 However,sequestration capacities depend on the metal and metalspeciation.28

The main aim of this work is the investigation of the specificiHg and MeHg pathways in a model fish, D. rerio using amultidisciplinary approach based on genetic biomarkers,electron microscopy, metallothionein concentration combinedwith complementary Hg speciation and isotopic signatureanalyses.29 An experimental approach was set up with adultzebrafish dietary contaminated with 10 000 ng Hg g−1 dryweight (dw) MeHg and iHg for two months. This Hg level issimilar to previous studies20,22,30,31 and near the concentrationin fish consumed by the French Guiana Amerindianpopulation.32 The assessment of iHg toxicity in fish in theliterature is sparse; simultaneous comparison of MeHg and iHgtoxicity even more so. Zebrafish provide a useful model tostudy contaminant effects and to elucidate mechanisms oftoxicity.33 Analyses were performed at different kinetic points(0, 7, 25, and 62 days) on skeletal muscle (consumed byhumans), liver (the major organ involved in metal detox-ification),34 and brain (major target of MeHg).7 Hg speciation,total Hg concentrations and metallothionein (MT) quantifica-tion were followed in the three organs at each sampling point.Genetic responses were investigated on 18 genes that encodefor proteins involved in key metabolic pathways of the cell:apoptosis (bax, p53), oxidative stress response (gpx4a, sod,sodmt), DNA repair (rad51), cellular detoxification (tap, mt2,hsf1), mitochondrial metabolism (vdac2, cox1, 12s), proteintransport (laptm4), nervous transmission (gfap, slc1a, ache), andlipid metabolism (hsd3, apoeb). In addition to these analyses,muscle and liver samples were evaluated by electronmicroscopy at 7 and 62 days to determine any histologicalchanges.

■ EXPERIMENTAL SECTIONExperimental Design. All procedures were approved by

the Aquitaine ethics committee for fish and birds (France,approval number 00493.01). Adult zebrafish from the same line

(EGRF7 line; body weight = 0.6 ± 0.1 g wet weight (ww);standard length = 30 ± 2 mm; n = 290, low-MeHg condition)were exposed to three dietary conditions for 62 days: 1/commercial fish food prepared from bycatch marine products,called low-MeHg (60 ± 10 ng Hg. g−1; 80% MeHg); 2/MeHgenriched food (11 580 ± 450 ng Hg. g−1; 95% MeHg); 3/iHgenriched food (11 920 ± 540 ng Hg. g−1; 1% MeHg). Thetheoretical quantity of mercury absorbed per fish throughoutthe experiment was evaluated in Table 1S. To minimize fishcontamination by the water, 1/2 of the water volume from eachtank was changed every day and tank bottoms were cleanedevery day to eliminate fish feces. Food preparation and moredetail about experimental design are described elsewhere.20

Fish were removed after 0, 7, 25, and 62 days. Skeletalmuscle, brain and liver were independently harvested. Samplesfor genetic analysis were put in RNA later (Qiagen) and storedat −80 °C until analysis. Samples for MT analysis wereimmediately placed under nitrogen atmosphere and stored at−80 °C. Samples for Hg analysis were stored at −20 °C. Formicroscopy, different organs/tissues were immediately im-mersed in a fixing solution.

GC-ICP-MS Analysis. All the samples (muscle, brain, liverand food) were digested with TMAH (Tetramethylammoniumhydroxide) in an analytical microwave and analyzed by GC-ICP-MS as detailed elsewhere.35,36 Quantification of Hg specieswas carried out by species specific isotope dilution, by addingthe appropriate amount of isotopically enriched Hg standards(199iHg and 201MeHg), and by applying isotope patterndeconvolution for data processing.36

Total RNA Extraction and Reverse Transcription ofTotal RNA. Total RNAs were extracted from 5 to 30 mg offresh tissue using the SV Total RNA Isolation System kit(Promega) according to manufacturer’s instructions. For eachexposure condition and for each organ, five replicates wereperformed. First-strand cDNA was synthesized from total RNA(3 μg) using the GoScript Reverse Transcription System kit(Promega). The cDNA mixture was kept at −20 °C untilrequired for the real-time PCR reaction.

Quantitative RT-PCR. The function, accession number andprimer pairs of the 18 genes and 3 reference genes used in ourstudy are listed in Table 2S. For each gene, the specific primerpairs were determined using the LightCycler probe designsoftware (version 1.0, Roche). The amplification of cDNA wasmonitored using the DNA intercalating dye SyberGreen I. Real-time PCR reactions were performed in a Stratagene MX3000PQPCR System (Agilent). Relative quantification of each geneexpression level was normalized according to the average ofthree housekeeping gene expression levels (β-actin, rpl13a, andEEF1A1). Relative mRNA expression of a gene was generatedusing the 2−ΔCT method.37 The mRNA induction factor (IF) ofeach gene in comparison with the control corresponds to thefollowing equation: IF = 2−ΔCT (Treatment)/2−ΔCT (control).The amplification program consisted of one cycle at 95 °C for10 min and 45 amplification cycles at 95 °C for 30 s, 55 °C for30 s, and 72 °C for 30 s.

Metallothionein Quantification. The concentration oftotal metallothionein protein (MT) was determined in skeletalmuscle, liver and brain of the zebrafish by mercury-saturationassay, using cold inorganic mercury.38,39 MT analyses wereconducted on 5 replicates per exposure condition, thesaturation assay being repeated twice per sample. MTconcentration was evaluated by flameless atomic absorptionspectrometry (AMA 254, Prague, Czech Republic). The exact

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DOI: 10.1021/acs.est.5b03586Environ. Sci. Technol. 2015, 49, 14560−14569

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quantity of Hg binding sites per MT molecule being unknownfor this species, MT levels were expressed in nmol Hg bindingsites g−1 ww. The measure of MT concentrations in relation tospecific tissue weight allowed us to directly compare MTconcentrations with bioaccumulation results expressed inrelation to tissue weight.Electron Microscopy. Muscle and liver of zebrafish were

completely sampled. Details about preparation of samples areavailable elsewhere.30 Briefly, different organs/tissues wereimmediately immersed in a fixing solution (2% glutaraldehydebuffered with 0.1 mmol/L sodium cacodylate solution, pH 7.4;osmolarity 420 mosmol/L) for 18 h at 4 °C. Ultrafine sections(500−700 Å) were placed on grids and further observed undera MET FEi TECNAI 12.Statistical Analysis. For each tissue/organ, the effect of

time and Hg exposure (independent variables) were performedby Factorial ANOVA after checking assumptions of normalityand homoscedasticity of the error term. If the assumption wasmet, the parametric Fisher’s Least Significant Difference (LSD)test was applied. If the assumption was not met, log, and box-cox data transformations40 were used, or a Kruskall−Wallis test.Comparisons of gene expression (dependent variables) wereperformed using a two-way ANOVA. When the assumptionwas not met after log transformation, the nonparametricMann−Whitney U test was used. In each test, p < 0.05 wasconsidered significant. All statistical investigations were

performed using STATISCA version 6.1 software (Statsoft,USA).

■ RESULTSFish Health. During the experiment, no fish mortality was

observed in any conditions. Fish appeared healthy on externalinspection (no injury, no fungoid growth, well-contrasted skincolors). No alteration of animals’ motility was observed. Theonly abnormal behavior observed was a decrease in fish’sappetite during the last 15 days of the experiment in iHg andMeHg conditions (i.e., food recovered at the bottom of theincubation units). Hg levels in the rest of the food and feces atthe bottom of units were measured and detailed in Feng et al.29

Kinetics of Hg Bioaccumulation in Zebrafish. Hgspeciation (Figure 1) and THg concentrations (Figure 1S)were determined in skeletal muscle, liver, and brain of zebrafishat 0, 7, 25, and 62 days in the low-MeHg, MeHg, and iHgconditions. MeHg concentrations in fish organs from the low-MeHg condition remained very low (maximum value = 536 ±65 ng Hg g−1 dw in muscle at 62 days) and relatively constant,even if a slight increase was observed (linked to the increase ofHg concentrations in water, see Feng et al.29).In the skeletal muscle, MeHg bioaccumulation increased

linearly during the first 25 days for the MeHg condition andthen slowed giving a final concentration of 31 869 ± 1 638 ngHg g−1 dw. In the iHg condition, even if the bioaccumulationwas very low, the iHg concentrations were statistically higher at

Figure 1. Hg speciation in the skeletal muscle, liver and brain of Danio rerio after 0, 7, 25, and 62 days of dietary exposure to iHg and MeHg.



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25 and 62 days against the low-MeHg condition, with aconstant linear increase. At 62 days, iHg and MeHg

concentrations were similar in muscle exposed to dietary iHg.iHg bioaccumulation was 76 fold lower in the iHg condition

Table 1. Significant Variations in Gene Expression As Compared to Low-MeHg Condition (Induction “x” or Repression Factors“/”) in Skeletal Muscle, Liver, and Brain from Danio rerio after 7, 25, and 62 Days of Dietary Exposure to MeHg and iHg (171ng fish−1 day−1)

Figure 2.Metallothionein concentrations in skeletal muscle (A), liver (B), and brain (C) of Danio rerio after 7, 25, and 62 days of dietary exposure to55 ng Hg g−1 dw (low-MeHg), 11 918 ng Hg g−1 dw (iHg), and 11 580 ng Hg g−1 dw (MeHg). Error bars represent standard errors, n = 3 to 5.Letters indicate statistical differences (p < 0.05).



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than MeHg bioaccumulation in the MeHg condition at 62 days.After MeHg exposure, bioaccumulation rate in the liver washigher than in muscle with a final bioaccumulation of 60 591 ±10 005 ng Hg g−1 dw at 62 days. iHg bioaccumulation at 62days was 17-fold lower in the liver (iHg condition) than MeHgbioaccumulation (MeHg condition). A decrease in iHgconcentration was observed at 25 days (1 771 ± 245 ng Hgg−1 dw, iHg condition). The brain samples from MeHgexposure showed a strong, linear MeHg accumulationtendency, the greatest of the three organs, and was observedover the 62 days (110 653 ± 41 090 ng Hg g−1 dw at 62 days).In the iHg condition, iHg concentration at 62 days in brain was49 times lower than MeHg bioaccumulation in MeHg exposure(2 612 ± 140 ng Hg g−1 dw).Gene Expression Levels. Apart from the basal genetic

expression, 7 out of the 18 genes studied (p53, hsf1, sodmt, cox1,ache, hsd3, apoeb) did not show any significant change in theirexpression rate whatever the contaminant, the exposure time orthe tissue (Table 1). However, the other 11 genes showed asignificant modification of their expression level depending onHg speciation, tissue, and exposure time. Sod gene, which isinvolved in oxidative stress response, showed the highestresponses in different conditions and organs. In the iHgcondition, sod gene was repressed in muscle, liver and brain at 7days (13.5-, 5.9-, 9.6-fold, respectively) then induced at 62 days(19.1-, 5.5-, 11.3-fold, respectively). In the MeHg condition, arepression of this gene was observed at 7 days only in muscle(27.8-fold) and brain (23.2-fold). Gfap gene expression wasdecreased significantly at each time point in muscle and only at7 days in liver of fish exposed to MeHg. Liver response is 9 foldhigher than in muscle at the same time points. For fish exposed

to iHg, this gene was also repressed in muscle at 25 days onlybut was half that in the MeHg condition at 25 days. In theMeHg condition at 7 days, in addition to gfap, 4 other geneswere repressed: rad51, mt2, and laptm4 in the liver and 12s inthe brain. This last gene was induced at 25 days in muscle. Inthe iHg condition at 7 days, the expression level of mt2 gene inbrain was induced, unlike the tap gene, which was repressed.This last gene was also repressed at 25 days in muscle. Aninduction of the expression level of the gpx4a gene in musclewas also marked at 7 days. The only expression modification ofthe bax gene, involved in cell apoptosis, was observed at 62days in brain of fish fed with iHg contaminated food, with aninduction of 2.2-fold compared to the low-MeHg condition. Inthis condition and at this time, inductions of vdac2, slc1a, and12s genes, in addition to the sod gene, were observed in muscle.

Metallothionein Response. Metallothionein concentra-tions remained low and relatively constant in skeletal muscle offish from the low-MeHg condition throughout the experiment(Figure 2). In the MeHg condition, MT concentrationsincreased significantly at 7 and 62 days compared to the low-MeHg condition. In the iHg condition, the same trend wasobserved with a statistical difference at 62 days compared to thelow-MeHg condition. No effect was observed on MTconcentrations in muscle. In liver, no significant effect ofmetals or time was shown in MT response. In brain of low-MeHg fish, a variation of MT concentrations was observed overtime. In the MeHg condition, a significant increase in MTconcentrations in brain was observed at 25 and 62 dayscompared to the low-MeHg condition. In the iHg condition, anincrease in MT concentrations was also observed up to 25 daysand then remained stable.

Figure 3. Observation by electron microscopy of zebrafish skeletal muscle and liver exposed to dietary iHg and MeHg at 62 days. Legend muscle: M,mitochondria; F, muscle fiber, I, interfiber bundle. Legend liver: H, hepatocyte; N, nucleus; BC, bile canaliculus; l, lipids; L, lysosome/peroxisome;Ma, macrophage; Er, endoplasmic reticulum.



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Tissue Observation by Electron Microscopy. Figure 3represents skeletal muscle and liver sections observed byelectron microscopy of zebrafish for the three conditions at 62days (data at 7 days are shown in Figure 2S). In muscle, noalteration of tissue was observed in the iHg conditionthroughout the experiment, while a significant disorganizationappeared in muscle exposed to MeHg: a disorganization ofmyofibrils, leading to broken fibers and spaces between fiberbundles. Mitochondria were also severely affected by MeHg at62 days where a full internal dislocation was noted. Liverexposure to dietary iHg had shown tissue recovery at 62 dayswith intact mitochondria (whereas some of them were affectedat 7 days; see Figure 2S). Conversely, exposure to dietaryMeHg induced a significant disruption in hepatic tissue, whichappeared clearly nonhomogeneous at 62 days with distortedhepatocytes, loss of stored substances, and damagedendoplasmic reticulum.

■ DISCUSSIONMercury Species Specific Bioaccumulation Pathways.

Zebrafish exposed to dietary MeHg showed a significantincrease in Hg concentrations in the three organs studied andgenerally followed the same trend as obtained by Gonzalez etal.,20 with similar experimental conditions. Fish fed with dietaryiHg showed a significant Hg bioaccumulation rate in the threeorgans compared to low-MeHg but lower than in fish exposedto dietary MeHg. MeHg is known to cross biologicalmembranes easily and to have a more efficient uptake andtrophic transfer than iHg.41−43 MeHg is stored in skeletalmuscle, from where it is slowly excreted (half-life around 400days).32,44,45 In liver and brain, detoxification mechanisms seemto exist since MeHg can be transformed or sequestered into lesstoxic species.46−49 However, the exact details of such processesare unknown. Hg concentrations measured in feces of fishexposed to iHg were higher than those exposed to MeHg (Fenget al.29). Berntssen et al.50 measured Hg concentrations in liverand brain of Atlantic salmon (Salmo salar), that had beencontaminated with dietary MeHg for 4 months, to be twice andalmost 30 fold, respectively, lower than in our study, for similardietary exposure concentrations. In the same study, thecontamination of fish with dietary iHg indicated a bioaccumu-lation rate 8- and 30-fold lower in liver and brain than in ourstudy, for the same iHg concentration levels in food. Thesedifferences in bioaccumulation can probably be explained bythe metabolism of zebrafish, a tropical fish, having a highermetabolism than salmon, a cold-water fish. Another parameteris the quantity of food given, based on percentage of bodyweight, half as much in their study (1.6% of salmonbodyweight) compared to ours (3% of zebrafish bodyweight).Even so, in our study no increase in mortality wasdemonstrated for any exposure condition throughout theexperiment. Other studies reported the absence of outwardsigns of MeHg toxicity at our concentration level in fish.20,51,52

Even at higher levels of dietary MeHg exposure (55.5 mg Hgkg−1 dw), mortality of juvenile blackfish (Orthodon micro-lepidotus) was very low for the 70 days experiment.53 However,the decrease in fish appetite observed over the last 15 days ofthis experiment after MeHg and iHg exposure may indicatetoxic effects. This leads to different capacities of organisms andorgans to eliminate Hg species over time and to the differencein input property in the cell of different Hg species, as shownbelow.

Major Genetic Responses Due to Both MeHg and iHgin Muscle Tissue. The lowest MeHg and iHg concentrationsof the three organs were reported in muscle. MT seemed toplay a role in detoxification of iHg and MeHg since an increaseover time of MT concentrations was shown. iHg can besequestered by MT and MeHg induces MT production as anindirect response to the oxidative stress in the cell. However, noresponse of the mt2 gene was observed under either Hg dietarycondition, in agreement with Gonzalez et al.20 regarding MeHgexposure. The first hypothesis is that the gene was inducedbefore, by induction pulse. The second explanation is that thebalance between MT synthesis and degradation was in favor ofsynthesis, that is, protein degradation was slowed down, whichhappens when MT sequesters metals, especially in the case ofiHg, which has the strongest binding affinity for theseproteins,54 as demonstrated by the incubation of liver cytosolextract with an isotopically enriched species.55 As Ho et al.56

and Gonzalez et al.20 observed, gene responses to MeHg appeartissue specific, even if similarities were observed in some tissues.The same conclusion is also valid for iHg in our study and inBerntssen et al.50 No correlation was observed between Hgbioaccumulation rate and genetic response of a tissue. Forexample, the majority of genetic responses, whatever conditionand exposure time, were observed in muscle (13 genes with asignificant modification of expression in muscle, 7 in brain and6 in liver) whereas Hg concentration in this tissue was thelowest observed. The nervous transmission function was mainlyaffected through the slc1a gene in the iHg condition and thegfap gene in MeHg and iHg conditions. The gfap gene encodedfor the glial fibrillary acidic protein, an intermediate filamentprotein involved in the maintenance of cellular strength andshape, and in the operation of the blood-brain barrier, that is,through its overexpression in astrocytes when a problem isdetected. In the MeHg condition, repression of the gfap gene inmuscle reflected the strong impact of MeHg. This conclusionwas correlated with our electron microscopy results where asignificant deterioration of the muscle fiber structure and adegenerative process in mitochondria were observed, aspreviously reported by Cambier et al.30 and De OliveiraRibeiro et al.57 Other functions in muscle such as mitochondrialmetabolism (12s and vdac2 genes), oxidative stress response(gpx4a and sod genes) and detoxification process (tap gene)were principally disturbed by iHg, but also by MeHg in thisstudy. Cambier et al.22 used the SAGE technique to identify 60up-regulated genes and 15 down-regulated genes involved inseveral functions (mitochondrial metabolism, detoxification,and general stress response or protein synthesis) in muscle ofzebrafish exposed to a diet containing 13.5 ng MeHg g−1. Therepression of the sod gene at 7 days in all fish tissue exposed todietary MeHg and iHg, except in liver-MeHg, was incontradiction with Gonzalez et al.:20 they observed aninduction of the sod gene at 7 and 21 days in muscle andliver of zebrafish with a diet containing 13.5 mg MeHg kg−1.However, a late induction of the sod gene (62 days) in the iHgcondition was observed in all tissues in our study. In the sameway, an up-regulation of the vdac2 and gpx4a genes in zebrafishmuscle exposed to MeHg was observed,20 whereas in this study,both genes were induced only in the iHg condition. Our resultsseem to indicate an early toxic impact of these two Hg specieson the redox defense system and, in addition, a later toxicimpact of iHg. However, here, only exposure to MeHg seems toinduce significant tissue damages.



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High Toxicity of MeHg in Liver Tissue. MeHgconcentrations in zebrafish liver exposed to dietary MeHgincreased over time, unlike results by Gonzalez et al.20 whoobserved a plateau from 7 days showing a demethylationprocess was in place (MeHg represented 66% of mercury at T0and then decreased to 36% after 63 days of exposure). Liver is adetoxification organ, able to accumulate high Hg concen-trations, demethylate MeHg to reduce its toxicity and tofacilitate its excretion.23 In this study, no demethylation processwas observed. Recently, it has been demonstrated using stableisotopically enriched tracers, that a reduction of the MeHgpercentage in Tilapia (Oreochromis niloticus) liver is not a resultof MeHg demethylation, but of transport of this species fromthe liver to other organs.58 Here, the absolute matches of theHg isotopic signature in liver and the supplied food confirmedliver as the target organ of ingested iHg (Feng et al.29). In theliver of zebrafish fed with an iHg diet, an increase in iHgconcentrations was observed at 7 days followed by a decreaseup to 25 days, suggesting organ redistribution of iHg. Thismatches the mass dependent isotopic pattern (i.e., expressed asδ202Hg) observed in different organs (Feng et al.29). Theprogressive enrichment of liver in heavier Hg isotopes (MDF,Mass Dependent Fractionation, from −0.8 to −0.2 ‰) after 7days could be associated with iHg redistribution to otherorgans, such as the brain. MT can bind iHg as observed in theliver of marine mammals.15 However, MT concentrations didnot increase over time whereas iHg concentrations are higherthan in muscle. This could be explained by the higher liver MTbasal level compared to other organs, showing thatdetoxification mechanisms are already active and higher iHgconcentrations are needed to induce MT synthesis.59,60

Repression of genes involved in DNA repair (rad51), cellulardetoxification (mt2), and response to oxidative stress (sod),nervous transmission (gfap) and protein transport (laptm4) at 7days suggest significant early toxic cellular damage by MeHg.These results contrast with Gonzalez et al.20 who, with similarexperimental conditions, noted induction of several genesinvolved in liver cell vital functions between 21 and 63 days,reflecting a severe toxic effect in hepatic cells. Ho et al.56 alsoobserved an up-regulation of antioxidant genes in the liver ofzebrafish embryos exposed from 48 to 72 hpf (hours postfertilization) to 60 μg L−1 MeHg in water, showing an oxidativestress and thus hepatocellular damage. Observation of liversections, contaminated with dietary MeHg, by electronmicroscopy showed significant damage to hepatic tissues. iHgexposure caused few responses (sod gene) showing the easierelimination of this pollutant by this detoxification organ helpedby a higher basal level of some genes (gpx4a) involved in theoxidative stress response. The absence of modification of thegenic expression after 7 days could be interpreted as anadaptive response of the redox defense system in the liver.Observation by electron microscopy showed tissue recoveryafter 62 days of iHg exposure. These results are in agreementwith De Oliveira Ribeiro et al.61 who showed that waterborneand trophic exposition of arctic charr to iHg had few effects onhepatic tissue. On the contrary, dietary MeHg caused variousdamage such as necrosis and disorganization in cytoplasm ofthe arctic charr (Salvelinus alpinus), despite the lowcontamination.High MeHg and iHg Bioaccumulation in Brain Tissue

Induced Metallothioneins. The highest Hg bioaccumulationrate was observed in the brain in the MeHg condition with aconstant increase of MeHg concentration over the 62 days of

the experiment. This kinetic of bioaccumulation correlates withMT concentrations (correlation coefficient = 99%). However,as in muscle, expression of the mt2 gene was not modified inthis condition whereas this gene encodes for a metallothioneinisoform known to sequester Hg. Assumptions for this result arethe same as those for muscle (induction by pulse of mt2, MTsynthesis higher than MT degradation). Several studies haveshown that MeHg exposure does not induce MT expression inthe brain of mammals or fish exposed to MeHg whereas iHg isan efficient inducer of MT.6,20,24,62 In brain, another isoform ofthis protein (mt3) is present, specific to this organ andconstitutively expressed63 and perhaps responsible for the MTconcentrations observed in our study. However, artificiallyincreasing brain MT protein levels protects against MeHgneurotoxicity.62 Dietary MeHg in zebrafish brain caused adown-regulation of genes involved in the oxidative stressresponse (sod) and mitochondrial metabolism (12s) at 7 days,demonstrating the early impact of MeHg in this organ, as weobserved for muscle and liver. The isotopic results (Feng etal.29) show a rapid isotopic re-equilibration (∼1.2 ‰ δ202Hgand Δ199Hg at 7 days) of the internal organs to the new MeHg-food source as a consequence of the high bioaccumulation rateof this organomercurial species. The only other decrease inSOD activity was in the brain tissue of Atlantic salmon fed foodcontaminated with 10 mg MeHg kg−1, whereas activity of thisenzyme increased in liver and kidney.50 In the iHg condition,iHg concentrations in brain increased linearly with similar finaliHg concentrations in the liver at 62 days. A significant increasein MT concentrations between 7 and 25 days followed by aplateau, and an induction of the mt2 gene at 7 days (2.5 fold)showed the brain’s ability to establish a cellular defensemechanism against iHg. At 7 days, iHg generated, as MeHg, amodification of the sod gene expression (oxidative stressresponse) but it also impacted genes involved in cellulardetoxification (tap, mt2). Oxidative stress (sod) and apoptosis(bax) functions were also induced at 62 days showing thesensitivity of the brain to iHg. Wang et al.64 exposed medaka(Oryzias melastigma) to acute concentrations of iHg and usedproteomic to show, in both liver and brain, an alteration ofsome proteins involved in the oxidative stress response,cytoskeletal assembly and signal transduction. Unlike Cambieret al.31 and Farkas et al.,65 the gfap gene was not induced in thezebrafish brain exposed to MeHg and iHg. The lack of responseof nervous transmission genes can be explained by their highbasal level of expression in brain. The low genetic response ofbrain compared to muscle, whereas this organ is known to bemore impacted by Hg, could be explained by the highspecificity of each region of the brain involving differentsensitivities and thus different genetic responses. The wholebrain analysis probably masked some results specific to a brainregion.66 No changes were observed in genetic expression of 13genes chosen in whole brain of zebrafish.20 On the contrary,other studies demonstrated effects of acute MeHg concen-trations on zebrafish brain exposed via water56 or viaintraperitoneal injection,24 with up-regulation of genes involvedin oxidative stress response and apoptosis.The influence of Hg species specific response on biomarkers

in fish organs reflected the dissimilar metabolization of both Hgspecies. In this study, zebrafish were the seventh descent of azebrafish line (breeding lab), probably with a poor geneticheritage compared to wild types. Differences with results ofother similar studies20,22,30,31 on the same species, such asgenetic responses in muscle and liver, may be explained by the



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origin of fish. Wild fish are more representative of whathappens in the environment and have far greater geneticvariability. Fish behavior may not be the same, that is, wild fishare not used to be fed daily, unlike laboratory fish. This couldhave an impact on biological functions. Thus, the origin of thefish used to conduct relevant ecotoxicological studies isquestioned and deserves reflection for future works.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.est.5b03586.

Details on experimental design, theoretical quantity ofmercury absorbed per fish during the experiment, averagetotal mercury (THg) concentrations in the skeletalmuscle, liver, and brain of Danio rerio, function, accessionnumbers, and specific primer matching with Danio reriogenes used for quantitative RT-PCR, differential geneexpressions, observation by electron microscopy ofzebrafish skeletal muscle and liver (PDF)

■ AUTHOR INFORMATIONCorresponding Author*Phone: + (33) 05 56 22 39 21. Fax: +(33) 05 56 54 93 83. E-mail: [email protected] authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was support by the French National ResearchAgency (ANR-11-CESA-0013, RIMNES project) and theCluster of Excellence COTE (ANR-10-LABX-45). The authorsacknowledge the Bordeaux Imaging Center (BIC, University ofBordeaux, UMS 3420 CNRS-US4 INSERM).

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Specific Effects of Dietary Methylmercury and Inorganic Mercury in Zebrafish (Danio rerio) Determined by Genetic, Histological, and Metallothionein Responses