Specific Effects of Dietary Methylmercury and Inorganic Mercury in

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Specific effects of dietary methylmercury and inorganic mercury in zebrafish (Danio rerio) determined by genetic, histological and metallothionein responses. Sophie Gentès, Regine Maury-Brachet, Caiyan Feng, Zoyne Pedrero, Emmanuel Tessier, Alexia Legeay, Nathalie Mesmer-Dudons, Magalie Baudrimont, Laurence Maurice, David Amouroux, and Patrice Gonzalez Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03586 • Publication Date (Web): 28 Oct 2015 Downloaded from http://pubs.acs.org on November 2, 2015

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Environmental Science & Technology

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Specific effects of dietary methylmercury and inorganic mercury in zebrafish (Danio

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rerio) determined by genetic, histological and metallothionein responses.

3

4

Sophie Gentès

†,

Régine Maury-Brachet

5

Tessier ǁ, Alexia Legeay

6

Maurice ⱡ§, David Amouroux ǁ, Patrice Gonzalez *‡.

†,

†,

Caiyan Feng ǁ, Zoyne Pedrero ǁ, Emmanuel

Nathalie Mesmer-Dudons

†,

Magalie Baudrimont

†,

Laurence

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Université de Bordeaux, EPOC, UMR CNRS 5805, Place du Dr B. Peyneau F-33120

8

†

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Arcachon, France.

10

‡

CNRS, EPOC, UMR 5805, F-33120 Arcachon, France.

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ǁ Laboratoire de Chimie Analytique, Bio-Inorganique et Environnement, Institut des Sciences

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Analytiques et de Physico-Chimie pour l’Environnement et les Matériaux (IPREM), CNRS-

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UPPA-UMR-5254, Hélioparc, 2 Avenue du Président Pierre Angot, F-64053 Pau, France.

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§ Observatoire Midi-Pyrénées, Laboratoire de Geosciences Environnement Toulouse,

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Université Paul Sabatier Toulouse III, 14 avenue Edouard Belin, 31400 Toulouse, France.

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ⱡ GET, IRD, F-31400 Toulouse, France.

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*Corresponding author: Patrice Gonzalez. Phone: + (33) 05 56 22 39 21; Fax: +(33) 05 56 54

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93 83; e-mail: [email protected]

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21

22

1 ACS Paragon Plus Environment


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Abstract

24 25

A multidisciplinary approach is proposed here to compare toxicity mechanisms of

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methylmercury (MeHg) and inorganic mercury (iHg) in muscle, liver and brain from

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zebrafish (Danio rerio). Animals were dietary exposed to (1) 50 ng Hg. g-1, 80% as MeHg;

28

(2) diet enriched in MeHg 10000 ng Hg. g-1, 95% as MeHg; (3) diet enriched in iHg 10000

29

ng Hg. g-1, 99% as iHg, for two months. Hg species specific bioaccumulation pathways were

30

highlighted, with a preferential bioaccumulation of MeHg in brain and iHg in liver. In the

31

same way, differences in genetic pattern were observed for both Hg species, (an early genetic

32

response (7 days) for both species in the three organs and a late genetic response (62 days) for

33

iHg) and revealed a dissimilar metabolization of both Hg species. Among the 18 studied

34

genes involved in key metabolic pathways of the cell, major genetic responses were observed

35

in muscle. Electron microscopy revealed damage mainly due to MeHg in muscle and also in

36

liver tissue. In brain, high MeHg and iHg concentrations induced metallothionein production.

37

Finally, the importance of the fish origin in ecotoxicological studies, here the 7th descent of a

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zebrafish line, is discussed.

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Keywords: mercury (Hg), toxicity, speciation, fish, genetic, metallothionein, microscopy.

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Introduction

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Mercury (Hg) is a toxic trace metal naturally present in different terrestrial

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compartments but human activities are clearly the major source of its remobilization in the

50

environment1-2. Exposure and uptake of Hg in aquatic organisms occurs mainly through food3-

51

4

52

contamination of aquatic ecosystems5.

53

Chemical speciation of Hg defines its absorption, assimilation, distribution and toxicity within

54

an organism6. Methylmercury (MeHg) is the most toxic species of Hg due to its ability to

55

cross biological membranes: it forms a complex with cysteine and enters the cell using neutral

56

amino acid carriers7. Another characteristic of MeHg is its capacity for biomagnification

57

along foodwebs8-9. In humans, the absorption efficiency of MeHg is 95 % regardless of the

58

route of exposure and about 10 % for inorganic Hg (iHg). While crossing biological barriers

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with difficulty, iHg still remains toxic, but at higher concentrations. Absorbed MeHg is

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assimilated through the gastrointestinal barrier, then passes into the blood where it is

61

distributed into target organs, mainly brain and muscle7. Chronic consumption of MeHg-

62

contaminated fish can cause adverse health effects in humans, especially on the central

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nervous and immune systems10-11. In contrast, iHg tends to be accumulated in detoxification

64

organs (liver and kidney) 12. Only a few studies have looked at iHg toxicity, although many

65

studies have been done on MeHg13. However, regulatory mechanisms, transport, metabolism

66

and elimination of Hg species in higher organisms such as mammals, including humans, are

67

still far from being fully explained6-7,14. Even though in recent years, the structural identity of

68

some bio-molecules containing Hg has been elucidated15-19, providing valuable information

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about its potential role in Hg detoxification, there is still a lack of knowledge of Hg pathways

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in living organisms. In cells, MeHg generates oxidative stress through reactive oxygen

. Therefore, the ichthyofauna represents a significant source of Hg for humans due to the



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species (ROS) formation. It interacts with cysteine sulfhydryl groups, disrupts calcium ion

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homeostasis, causes damage such as mitochondrial disturbance, lipid peroxidation, DNA

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damage, membrane structure alteration, cell cycle dysfunction and apoptosis, damage to the

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immune system and glucose metabolism disturbance6. Several studies have observed these

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changes through transcriptome study of adult zebrafish (Danio rerio) and fathead minnow

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(Pimephales promelas) (two biological models) contaminated with different levels of MeHg

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(acute, sublethal, and chronic) and via the direct or the trophic pathways in muscle, brain and

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liver20-24. Cells also have anti-oxidant enzymes to fight against oxidative stress, like

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superoxide dismutase and glutathione peroxidase25. Other defense mechanisms exist such as

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active efflux pumps belonging to the family of ATP binding cassette (ABC) transporters26 or

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metal sequestration proteins like metallothioneins. These low molecular weight cysteine-rich

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proteins are able to chelate and scavenge metals27. However, sequestration capacities depend

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on the metal and metal speciation28.

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The main aim of this work is the investigation of the specific iHg and MeHg pathways in a

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model fish, D. rerio using a multidisciplinary approach based on genetic biomarkers, electron

86

microscopy, metallothionein concentration combined with complementary Hg speciation and

87

isotopic signature analyses (Feng et al. 2015, companion publication). An experimental

88

approach was set up with adult zebrafish dietary contaminated with 10 000 ng Hg. g-1 dry

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weight (dw) MeHg and iHg for two months. This Hg level is similar to previous

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studies20,22,30,31 and near the concentration in fish consumed by the French Guiana Amerindian

91

population32. The assessment of iHg toxicity in fish in the literature is sparse; simultaneous

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comparison of MeHg and iHg toxicity even more so. Zebrafish provide a useful model to

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study contaminant effects and to elucidate mechanisms of toxicity29. Analyses were

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performed at different kinetic points (0, 7, 25 and 62 days) on skeletal muscle (consumed by

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humans), liver (the major organ involved in metal detoxification)33, and brain (major target of 4 ACS Paragon Plus Environment

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MeHg)7. Hg speciation, total Hg concentrations and metallothionein (MT) quantification were

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followed in the three organs at each sampling point. Genetic responses were investigated on

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18 genes that encode for proteins involved in key metabolic pathways of the cell: apoptosis

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(bax, p53), oxidative stress response (gpx4a, sod, sodmt), DNA repair (rad51), cellular

100

detoxification (tap, mt2, hsf1), mitochondrial metabolism (vdac2, cox1, 12s), protein transport

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(laptm4), nervous transmission (gfap, slc1a, ache) and lipid metabolism (hsd3, apoeb). In

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addition to these analyses, muscle and liver samples were evaluated by electron microscopy at

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7 and 62 days to determine any histological changes.

104 105

Experimental

106 107

Experimental design

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All procedures were approved by the Aquitaine ethics committee for fish and birds (France,

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approval number: 00493.01). Adult zebrafish from the same line (EGRF7 line; body weight:

110

0.6 ± 0.1 g wet weight (ww); standard length: 30 ± 2 mm; n = 290, low-MeHg condition)

111

were exposed to three dietary conditions for 62 days: 1/ commercial fish food prepared from

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bycatch marine products, called low-MeHg (60 ± 10 ng Hg. g-1; 80 % MeHg); 2/ MeHg

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enriched food (11 580 ± 450 ng Hg. g-1; 95 % MeHg); 3/ iHg enriched food (11 920 ± 540 ng

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Hg. g-1; 1 % MeHg). The theoretical quantity of mercury absorbed per fish throughout the

115

experiment was evaluated in Table 1S. To minimize fish contamination by the water, ½ of the

116

water volume from each tank was changed every day and tank bottoms were cleaned every

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day to eliminate fish feces. Food preparation and more detail about experimental design are

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described elsewhere 20.

119

Fish were removed after 0, 7, 25 and 62 days. Skeletal muscle, brain and liver were

120

independently harvested. Samples for genetic analysis were put in RNA later (Qiagen) and



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stored at -80°C until analysis. Samples for MT analysis were immediately placed under

122

nitrogen atmosphere and stored at -80°C. Samples for Hg analysis were stored at -20°C. For

123

microscopy, different organs/tissues were immediately immersed in a fixing solution.

124 125

GC-ICP-MS analysis

126

All the

127

(Tetramethylammonium hydroxide) in an analytical microwave and analyzed by GC-ICP-MS

128

as detailed elsewhere

129

isotope dilution, by adding the appropriate amount of isotopically enriched Hg standards

130

(199iHg and 201MeHg, and by applying isotope pattern deconvolution for data processing35 ).

samples (muscle,

brain, liver and food) were digested with

TMAH

34, 35

. Quantification of Hg species was carried out by species specific

131 132

Total RNA extraction and reverse transcription of total RNA

133

Total RNAs were extracted from 5 to 30 mg of fresh tissue using the SV Total RNA Isolation

134

System kit (Promega) according to manufacturer’s instructions. For each exposure condition

135

and for each organ, five replicates were performed. First-strand cDNA was synthesized from

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total RNA (3 µg) using the GoScript Reverse Transcription System kit (Promega). The cDNA

137

mixture was kept at −20°C until required for the real-time PCR reaction.

138 139

Quantitative RT-PCR

140

The function, accession number and primer pairs of the 18 genes and 3 reference genes used

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in our study are listed in Table 2S. For each gene, the specific primer pairs were determined

142

using the LightCycler probe design software (version 1.0, Roche). The amplification of cDNA

143

was monitored using the DNA intercalating dye SyberGreen I. Real-time PCR reactions were

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performed in a Stratagene MX3000P QPCR System (Agilent). Relative quantification of each

145

gene expression level was normalized according to the average of three housekeeping gene


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expression levels (β- actin, rpl13a and EEF1A1). Relative mRNA expression of a gene was

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generated using the 2-∆CT method36. The mRNA induction factor (IF) of each gene in

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comparison with the control corresponds to the following equation: IF = 2-∆CT (Treatment) /

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2-∆CT (control). The amplification program consisted of one cycle at 95 °C for 10 min and 45

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amplification cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s.

151 152

Metallothionein quantification

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The concentration of total metallothionein protein (MT) was determined in skeletal muscle,

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liver and brain of the zebrafish by mercury-saturation assay, using cold inorganic

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mercury37,38. MT analyses were conducted on 5 replicates per exposure condition, the

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saturation assay being repeated twice per sample. MT concentration was evaluated by

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flameless atomic absorption spectrometry (AMA 254, Prague, Czech Republic). The exact

158

quantity of Hg binding sites per MT molecule being unknown for this species, MT levels

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were expressed in nmol Hg binding sites. g−1 ww. The measure of MT concentrations in

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relation to specific tissue weight allowed us to directly compare MT concentrations with

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bioaccumulation results expressed in relation to tissue weight.

162 163

Electron Microscopy

164

Muscle and liver of zebrafish were completely sampled. Details about preparation of samples

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are available elsewhere30. Briefly, different organs/tissues were immediately immersed in a

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fixing solution (2 % glutaraldehyde buffered with 0.1 mmol/L sodium cacodylate solution, pH

167

7.4; osmolarity 420 mosmol/L) for 18 h at 4 °C. Ultrafine sections (500 – 700 A°) were

168

placed on grids and further observed under a MET FEi TECNAI 12.

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Statistical analysis



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For each tissue/organ, the effect of time and Hg exposure (independent variables) were

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performed by Factorial ANOVA after checking assumptions of normality and

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homoscedasticity of the error term. If the assumption was met, the parametric Fisher's Least

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Significant Difference (LSD) test was applied. If the assumption was not met, log and box-

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cox data transformations39 were used, or a Kruskall-Wallis test. Comparisons of gene

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expression (dependent variables) were performed using a two-way ANOVA. When the

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assumption was not met after log transformation, the non-parametric Mann–Whitney U test

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was used. In each test, p < 0.05 was considered significant. All statistical investigations were

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

179 180

Results

181 182

Fish health

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During the experiment, no fish mortality was observed in any conditions. Fish appeared

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healthy on external inspection (no injury, no fungoid growth, well-contrasted skin colors). No

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alteration of animals’ motility was observed. The only abnormal behavior observed was a

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decrease in fish’s appetite during the last 15 days of the experiment in iHg and MeHg

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conditions (i.e. food recovered at the bottom of the incubation units). Hg levels in the rest of

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the food and feces at the bottom of units were measured and detailed in Feng et al.

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2015, companion publication.

190 191

Kinetics of Hg bioaccumulation in zebrafish

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Hg speciation (Figure 1) and THg concentrations (Figure 1S) were determined in skeletal

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muscle, liver and brain of zebrafish at 0, 7, 25 and 62 days in the low-MeHg, MeHg and iHg

194

conditions. MeHg concentrations in fish organs from the low-MeHg condition remained very


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low (maximum value: 536 ± 65 ng MeHg. g-1 dw in muscle at 62 days) and relatively

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constant, even if a slight increase was observed (linked to the increase of Hg concentrations in

197

water, see Feng et al. 2015, companion paper).

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In the skeletal muscle, MeHg bioaccumulation increased linearly during the first 25 days for

199

the MeHg condition and then slowed giving a final concentration of 31 869 ± 1 638 ng Hg. g-1

200

dw. In the iHg condition, even if the bioaccumulation was very low, the iHg concentrations

201

were statistically higher at 25 and 62 days against the low-MeHg condition, with a constant

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linear increase. At 62 days, iHg and MeHg concentrations were similar in muscle exposed to

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dietary iHg. iHg bioaccumulation was 76 fold lower in the iHg condition than MeHg

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bioaccumulation in the MeHg condition at 62 days. After MeHg exposure, bioaccumulation

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rate in the liver was higher than in muscle with a final bioaccumulation of 60 591 ± 10 005 ng

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Hg. g-1 dw at 62 days. iHg bioaccumulation at 62 days was 17 fold lower in the liver (iHg

207

condition) than MeHg bioaccumulation (MeHg condition). A decrease in iHg concentration

208

was observed at 25 days (1 771 ± 245 ng Hg. g-1 dw, iHg condition). The brain samples from

209

MeHg exposure showed a strong, linear MeHg accumulation tendency, the greatest of the

210

three organs, and was observed over the 62 days (110 653 ± 41 090 ng Hg. g-1 dw at 62 days).

211

In the iHg condition, iHg concentration at 62 days in brain was 49 times lower than MeHg

212

bioaccumulation in MeHg exposure (2 612 ± 140 ng Hg. g-1 dw).

213 214

Gene expression levels

215

Apart from the basal genetic expression, 7 out of the 18 genes studied (p53, hsf1, sodmt, cox1,

216

ache, hsd3, apoeb) did not show any significant change in their expression rate whatever the

217

contaminant, the exposure time or the tissue (Table 1). However, the other 11 genes showed a

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significant modification of their expression level depending on Hg speciation, tissue and

219

exposure time. Sod gene, which is involved in oxidative stress response, showed the highest



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responses in different conditions and organs. In the iHg condition, sod gene was repressed in

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muscle, liver and brain at 7 days (13.5 fold, 5.9 fold, 9.6 fold respectively) then induced at 62

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days (19.1 fold, 5.5 fold, 11.3 fold respectively). In the MeHg condition, a repression of this

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gene was observed at 7 days only in muscle (27.8 fold) and brain (23.2 fold). Gfap gene

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expression was decreased significantly at each time point in muscle and only at 7 days in liver

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of fish exposed to MeHg. Liver response is 9 fold higher than in muscle at the same time

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points. For fish exposed to iHg, this gene was also repressed in muscle at 25 days only, but

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was half that in the MeHg condition at 25 days. In the MeHg condition at 7 days, in addition

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to gfap, 4 other genes were repressed: rad51, mt2 and laptm4 in the liver and 12s in the brain.

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This last gene was induced at 25 days in muscle. In the iHg condition at 7 days, the

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expression level of mt2 gene in brain was induced, unlike the tap gene which was repressed.

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This last gene was also repressed at 25 days in muscle. An induction of the expression level of

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the gpx4a gene in muscle was also marked at 7 days. The only expression modification of the

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bax gene, involved in cell apoptosis, was observed at 62 days in brain of fish fed with iHg

234

contaminated food, with an induction of 2.2 fold compared to the low-MeHg condition. In this

235

condition and at this time, inductions of vdac2, slc1a and 12s genes, in addition to the sod

236

gene, were observed in muscle.

237 238

Metallothionein response

239

Metallothionein concentrations remained low and relatively constant in skeletal muscle of fish

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from the low-MeHg condition throughout the experiment (Figure 2). In the MeHg condition,

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MT concentrations increased significantly at 7 and 62 days compared to the low-MeHg

242

condition. In the iHg condition, the same trend was observed with a statistical difference at 62

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days compared to the low-MeHg condition. No effect was observed on MT concentrations in

244

muscle. In liver, no significant effect of metals or time was shown in MT response. In brain of


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low-MeHg fish, a variation of MT concentrations was observed over time. In the MeHg

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condition, a significant increase in MT concentrations in brain was observed at 25 and 62

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days compared to the low-MeHg condition. In the iHg condition, an increase in MT

248

concentrations was also observed up to 25 days and then remained stable.

249 250

Tissue observation by electron microscopy

251

Figure 3 represents skeletal muscle and liver sections observed by electron microscopy of

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zebrafish for the three conditions at 62 days (data at 7 days are shown in SI Figure 2S). In

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muscle, no alteration of tissue was observed in the iHg condition throughout the experiment,

254

while a significant disorganization appeared in muscle exposed to MeHg: a disorganization of

255

myofibrils, leading to broken fibers and spaces between fiber bundles. Mitochondria were

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also severely affected by MeHg at 62 days where a full internal dislocation was noted. Liver

257

exposure to dietary iHg had shown tissue recovery at 62 days with intact mitochondria

258

(whereas some of them were affected at 7 days - see SI Figure 2S). Conversely, exposure to

259

dietary MeHg induced a significant disruption in hepatic tissue, which appeared clearly

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nonhomogeneous at 62 days with distorted hepatocytes, loss of stored substances and

261

damaged endoplasmic reticulum.

262 263

Discussion

264 265

Mercury species specific bioaccumulation pathways

266

Zebrafish exposed to dietary MeHg showed a significant increase in Hg concentrations in the

267

three organs studied and generally followed the same trend as obtained by Gonzalez et al. 20,

268

with similar experimental conditions. Fish fed with dietary iHg showed a significant Hg

269

bioaccumulation rate in the three organs compared to low-MeHg but lower than in fish



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exposed to dietary MeHg. MeHg is known to cross biological membranes easily and to have a

271

more efficient uptake and trophic transfer than iHg40-42. MeHg is stored in skeletal muscle,

272

from where it is slowly excreted (half-life around 400 days)32,43,44. In liver and brain,

273

detoxification mechanisms seem to exist since MeHg can be transformed or sequestered into

274

less toxic species45-48. However, the exact details of such processes are unknown. Hg

275

concentrations measured in feces of fish exposed to iHg were higher than those exposed to

276

MeHg (Feng et al. 2015, companion publication). Berntssen et al.49 measured Hg

277

concentrations in liver and brain of Atlantic salmon (Salmo salar), that had been

278

contaminated with dietary MeHg for 4 months, to be twice and almost 30 fold, respectively,

279

lower than in our study, for similar dietary exposure concentrations. In the same study, the

280

contamination of fish with dietary iHg indicated a bioaccumulation rate 8 fold and 30 fold

281

lower in liver and brain than in our study, for the same iHg concentration levels in food.

282

These differences in bioaccumulation can probably be explained by the metabolism of

283

zebrafish, a tropical fish, having a higher metabolism than salmon, a cold-water fish. Another

284

parameter is the quantity of food given, based on percentage of body weight, half as much in

285

their study (1.6% of salmon bodyweight) compared to ours (3% of zebrafish bodyweight).

286

Even so, in our study no increase in mortality was demonstrated for any exposure condition

287

throughout the experiment. Other studies reported the absence of outward signs of MeHg

288

toxicity at our concentration level in fish20,50,51. Even at higher levels of dietary MeHg

289

exposure (55.5 mg Hg. kg-1 dw), mortality of juvenile blackfish (Orthodon microlepidotus)

290

was very low for the 70 days experiment52. However, the decrease in fish appetite observed

291

over the last 15 days of this experiment after MeHg and iHg exposure may indicate toxic

292

effects. This leads to different capacities of organisms, and organs, to eliminate Hg species

293

over time and to the difference in input property in the cell of different Hg species, as shown

294

below.


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Major genetic responses due to both MeHg and iHg in muscle tissue

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The lowest MeHg and iHg concentrations of the three organs were reported in muscle. MT

298

seemed to play a role in detoxification of iHg and MeHg since an increase over time of MT

299

concentrations was shown. iHg can be sequestered by MT and MeHg induces MT production

300

as an indirect response to the oxidative stress in the cell. However, no response of the mt2

301

gene was observed under either Hg dietary condition, in agreement with Gonzalez et al.20

302

regarding MeHg exposure. The first hypothesis is that the gene was induced before, by

303

induction pulse. The second explanation is that the balance between MT synthesis and

304

degradation was in favor of synthesis, i.e. protein degradation was slowed down, which

305

happens when MT sequesters metals, especially in the case of iHg which has the strongest

306

binding affinity for these proteins53, as demonstrated by the incubation of liver cytosol extract

307

with an isotopically enriched species54. As Ho et al. 56 and Gonzalez et al.20 observed, gene

308

responses to MeHg appears tissue specific, even if similarities were observed in some tissues.

309

The same conclusion is also valid for iHg in our study and in Berntssen et al.

310

correlation was observed between Hg bioaccumulation rate and genetic response of a tissue.

311

For example, the majority of genetic responses, whatever condition and exposure time, were

312

observed in muscle (13 genes with a significant modification of expression in muscle, 7 in

313

brain and 6 in liver) whereas Hg concentration in this tissue was the lowest observed. The

314

nervous transmission function was mainly affected through the slc1a gene in the iHg

315

condition and the gfap gene in MeHg and iHg conditions. The gfap gene, encoded for the glial

316

fibrillary acidic protein, an intermediate filament protein involved in the maintenance of

317

cellular strength and shape, and in the operation of the blood-brain barrier, i.e. through its

318

over-expression in astrocytes when a problem is detected. In the MeHg condition, repression

319

of the gfap gene in muscle reflected the strong impact of MeHg. This conclusion was

49

. No



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correlated with our electron microscopy results where a significant deterioration of the muscle

321

fiber structure and a degenerative process in mitochondria were observed, as previously

322

reported by Cambier et al. 30 and De Oliveira Ribeiro et al. 57. Other functions in muscle such

323

as mitochondrial metabolism (12s and vdac2 genes), oxidative stress response (gpx4a and sod

324

genes) and detoxification process (tap gene) were principally disturbed by iHg, but also by

325

MeHg in this study. Cambier et al. 22 used the SAGE technique to identify 60 up-regulated

326

genes and 15 down-regulated genes involved in several functions (mitochondrial metabolism,

327

detoxification, and general stress response or protein synthesis) in muscle of zebrafish

328

exposed to a diet containing 13.5 mg MeHg. kg-1. The repression of the sod gene at 7 days in

329

all fish tissue exposed to dietary MeHg and iHg, except in liver where MeHg was in

330

contradiction with Gonzalez et al.20: they observed an induction of the sod gene at 7 and 21

331

days in muscle and liver of zebrafish with a diet containing 13.5 mg MeHg.kg-1. However, a

332

late induction of the sod gene (62 days) in the iHg condition was observed in all tissues in our

333

study. In the same way, an up-regulation of the vdac2 and gpx4a genes in zebrafish muscle

334

exposed to MeHg was observed20, whereas in this study, both genes were induced only in the

335

iHg condition. Our results seem to indicate an early toxic impact of these two Hg species on

336

the redox defense system and, in addition, a later toxic impact of iHg. However, here, only

337

exposure to MeHg seems to induce significant tissue damages.

338 339

High toxicity of MeHg in liver tissue

340

MeHg concentrations in zebrafish liver exposed to dietary MeHg increased over time, unlike

341

results by Gonzalez et al. 20 who observed a plateau from 7 days showing a demethylation

342

process was in place (MeHg represented 66 % of mercury at T0 and then decreased to 36 %

343

after 63 days of exposure). Liver is a detoxification organ, able to accumulate high Hg

344

concentrations, demethylate MeHg to reduce its toxicity and to facilitate its excretion23. In this


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study, no demethylation process was observed. Recently, it has been demonstrated using

346

stable isotopically enriched tracers, that a reduction of the MeHg percentage in Tilapia

347

(Oreochromis niloticus) liver is not a result of MeHg demethylation, but of transport of this

348

species from the liver to other organs58. Here, the absolute matches of the Hg isotopic

349

signature in liver and the supplied food confirmed liver as the target organ of ingested iHg

350

(Feng et al. 2015, companion publication). In the liver of zebrafish fed with an iHg diet, an

351

increase in iHg concentrations was observed at 7 days followed by a decrease up to 25 days,

352

suggesting organ redistribution of iHg. This matches the mass dependent isotopic pattern (i.e.

353

expressed as δ202Hg) observed in different organs (Feng et al. 2015, companion publication).

354

The progressive enrichment of liver in heavier Hg isotopes (MDF, Mass Dependent

355

Fractionation, from -0.8 to -0.2 ‰) after 7 days could be associated with iHg redistribution to

356

other organs, such as the brain. MT can bind iHg as observed in the liver of marine

357

mammals15. However, MT concentrations did not increase over time whereas iHg

358

concentrations are higher than in muscle. This could be explained by the higher liver MT

359

basal level compared to other organs, showing that detoxification mechanisms are already

360

active and higher iHg concentrations are needed to induce MT synthesis

361

genes involved in DNA repair (rad51), cellular detoxification (mt2) and response to oxidative

362

stress (sod), nervous transmission (gfap) and protein transport (laptm4) at 7 days suggest

363

significant early toxic cellular damage by MeHg. These results contrast with Gonzalez et al.20

364

who, with similar experimental conditions, noted induction of several genes involved in liver

365

cell vital functions between 21 and 63 days, reflecting a severe toxic effect in hepatic cells.

366

Ho et al.

367

embryos exposed from 48 to 72 hpf (hours post fertilization) to 60 µg. L-1 MeHg in water,

368

showing an oxidative stress and thus hepatocellular damage. Observation of liver sections,

369

contaminated with dietary MeHg, by electron microscopy showed significant damage to

56

59-60

. Repression of

also observed an up-regulation of antioxidant genes in the liver of zebrafish



Page 16 of 35

370

hepatic tissues. iHg exposure caused few responses (sod gene) showing the easier elimination

371

of this pollutant by this detoxification organ helped by a higher basal level of some genes

372

(gpx4a) involved in the oxidative stress response. The absence of modification of the genic

373

expression after 7 days could be interpreted as an adaptive response of the redox defense

374

system in the liver. Observation by electron microscopy showed tissue recovery after 62 days

375

of iHg exposure. These results are in agreement with De Oliveira Ribeiro et al. 61 who showed

376

that waterborne and trophic exposition of arctic charr to iHg had few effects on hepatic tissue.

377

On the contrary, dietary MeHg caused various damage such as necrosis and disorganization in

378

cytoplasm of the arctic charr (Salvelinus alpinus), despite the low contamination.

379 380

High MeHg and iHg bioaccumulation in brain tissue induced metallothioneins

381

The highest Hg bioaccumulation rate was observed in the brain in the MeHg condition with a

382

constant increase of MeHg concentration over the 62 days of the experiment. This kinetic of

383

bioaccumulation correlates with MT concentrations (correlation coefficient = 99 %).

384

However, as in muscle, expression of the mt2 gene was not modified in this condition whereas

385

this gene encodes for a metallothionein isoform known to sequester Hg. Assumptions for this

386

result are the same as those for muscle (induction by pulse of mt2, MT synthesis higher than

387

MT degradation). Several studies have shown that MeHg exposure does not induce MT

388

expression in the brain of mammals or fish exposed to MeHg whereas iHg is an efficient

389

inducer of MT6,20,24,62 . In brain, another isoform of this protein (mt3) is present, specific to

390

this organ and constitutively expressed63 and perhaps responsible for the MT concentrations

391

observed in our study. However, artificially increasing brain MT protein levels protects

392

against MeHg neurotoxicity62. Dietary MeHg in zebrafish brain caused a down-regulation of

393

genes involved in the oxidative stress response (sod) and mitochondrial metabolism (12s) at 7

394

days, demonstrating the early impact of MeHg in this organ, as we observed for muscle and


Page 17 of 35


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liver. The isotopic results (Feng et al. 2015, companion publication) show a rapid isotopic re-

396

equilibration (~1.2 ‰ δ202Hg and ∆199Hg at 7 days) of the internal organs to the new MeHg-

397

food source as a consequence of the high bioaccumulation rate of this organomercurial

398

species. The only other decrease in SOD activity was in the brain tissue of Atlantic salmon

399

fed food contaminated with 10 mg MeHg. kg-1, whereas activity of this enzyme increased in

400

liver and kidney49. In the iHg condition, iHg concentrations in brain increased linearly with

401

similar final iHg concentrations in the liver at 62 days. A significant increase in MT

402

concentrations between 7 and 25 days followed by a plateau, and an induction of the mt2 gene

403

at 7 days (2.5 fold) showed the brain's ability to establish a cellular defense mechanism

404

against iHg. At 7 days, iHg generated, as MeHg, a modification of the sod gene expression

405

(oxidative stress response) but it also impacted genes involved in cellular detoxification (tap,

406

mt2). Oxidative stress (sod) and apoptosis (bax) functions were also induced at 62 days

407

showing the sensitivity of the brain to iHg.

408

melastigma) to acute concentrations of iHg and used proteomic to show, in both liver and

409

brain, an alteration of some proteins involved in the oxidative stress response, cytoskeletal

410

assembly and signal transduction. Unlike Cambier et al. 31 and Farkas et al. 65, the gfap gene

411

was not induced in the zebrafish brain exposed to MeHg and iHg. The lack of response of

412

nervous transmission genes can be explained by their high basal level of expression in brain.

413

The low genetic response of brain compared to muscle, whereas this organ is known to be

414

more impacted by Hg, could be explained by the high specificity of each region of the brain

415

involving different sensitivities and thus different genetic responses. The whole brain analysis

416

probably masked some results specific to a brain region66. No changes were observed in

417

genetic expression of 13 genes chosen in whole brain of zebrafish20. On the contrary, other

418

studies demonstrated effects of acute MeHg concentrations on zebrafish brain exposed via

Wang et al.

64

exposed medaka (Oryzias



Page 18 of 35

419

water56 or via intraperitoneal injection24, with up-regulation of genes involved in oxidative

420

stress response and apoptosis.

421

The influence of Hg species specific response on biomarkers in fish organs reflected the

422

dissimilar metabolization of both Hg species. In this study, zebrafish were the 7th descent of a

423

zebrafish line (breeding lab), probably with a poor genetic heritage compared to wild types.

424

Differences with results of other similar studies20,22,30,31 on the same species, such as genetic

425

responses in muscle and liver, may be explained by the origin of fish. Wild fish are more

426

representative of what happens in the environment and have far greater genetic variability.

427

Fish behavior may not be the same, i.e. wild fish are not used to be fed daily, unlike

428

laboratory fish. This could have an impact on biological functions. Thus the origin of the fish

429

used to conduct relevant ecotoxicological studies is questioned and deserves reflection for

430

future works.

431 432

Acknowledgments

433

This work was support by the French National Research Agency (ANR-11-CESA-0013,

434

RIMNES project) and the Cluster of Excellence COTE (ANR-10-LABX-45). The authors

435

acknowledge the Bordeaux Imaging Center (BIC- University of Bordeaux, UMS 3420 CNRS

436

- US4 INSERM).

437

Legend to figures

438

Figure. TOC abstract art (graphic for manuscript).

439

Figure 1. Hg speciation in the skeletal muscle, liver and brain of Danio rerio after 0, 7, 25 and

440

62 days of dietary exposure to iHg and MeHg.


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Table 1. Significant variations in gene expression as compared to the low-MeHg condition

442

(induction: “x” or repression factors: “/”) in skeletal muscle, liver and brain from Danio rerio

443

after 7, 25 and 62 days of dietary exposure to MeHg and iHg (171 ng. fish-1. day-1).

444

Figure 2. Metallothionein concentrations in skeletal muscle (A), liver (B) and brain (C) of

445

Danio rerio after 7, 25 and 62 days of dietary exposure to 55 ng Hg.g-1dw (low-MeHg), 11

446

918 ng Hg.g-1dw (iHg), and 11 580 ng Hg.g-1dw (MeHg). Error bars represent standard

447

errors, n = 3 to 5. Letters indicate statistical differences (p

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