Feeding mice with diets containing mercury-contaminated fish flesh from French Guiana: a model for the mercurial intoxication of the Wayana Amerindians
- Jean-Paul Bourdineaud1Email author,
- Nadège Bellance2,
- Giovani Bénard2,
- Daniel Brèthes3,
- Masatake Fujimura4,
- Patrice Gonzalez1,
- Aline Marighetto5,
- Régine Maury-Brachet1,
- Cécile Mormède5,
- Vanessa Pédron1,
- Jean-Nicolas Philippin5,
- Rodrigue Rossignol2,
- William Rostène6,
- Masumi Sawada4 and
- Muriel Laclau1, 3, 5
© Bourdineaud et al; licensee BioMed Central Ltd. 2008
Received: 19 February 2008
Accepted: 29 October 2008
Published: 29 October 2008
In 2005, 84% of Wayana Amerindians living in the upper marshes of the Maroni River in French Guiana presented a hair mercury concentration exceeding the limit set up by the World Health Organization (10 μg/g). To determine whether this mercurial contamination was harmful, mice have been fed diets prepared by incorporation of mercury-polluted fish from French Guiana.
Four diets containing 0, 0.1, 1, and 7.5% fish flesh, representing 0, 5, 62, and 520 ng methylmercury per g, respectively, were given to four groups of mice for a month. The lowest fish regimen led to a mercurial contamination pressure of 1 ng mercury per day per g of body weight, which is precisely that affecting the Wayana Amerindians.
The expression of several genes was modified with mercury intoxication in liver, kidneys, and hippocampus, even at the lowest tested fish regimen. A net genetic response could be observed for mercury concentrations accumulated within tissues as weak as 0.15 ppm in the liver, 1.4 ppm in the kidneys, and 0.4 ppm in the hippocampus. This last value is in the range of the mercury concentrations found in the brains of chronically exposed patients in the Minamata region or in brains from heavy fish consumers. Mitochondrial respiratory rates showed a 35–40% decrease in respiration for the three contaminated mice groups. In the muscles of mice fed the lightest fish-containing diet, cytochrome c oxidase activity was decreased to 45% of that of the control muscles. When mice behavior was assessed in a cross maze, those fed the lowest and mid-level fish-containing diets developed higher anxiety state behaviors compared to mice fed with control diet.
We conclude that a vegetarian diet containing as little as 0.1% of mercury-contaminated fish is able to trigger in mice, after only one month of exposure, disorders presenting all the hallmarks of mercurial contamination.
Methylmercury is a neurotoxic compound, which has been shown to be the cause of the Minamata disease. Diseased persons were struck by ataxia and suffered from visual, sensorial and hearing problems, seizures, memory disabilities, muscular weakness and cramps . The effects of low amplitude prenatal exposure on neurological development have been described, and human exposure to methylmercury has been linked to fish and shellfish consumption .
In French Guiana, clandestine gold mining contaminates numerous sites, both terrestrial and aquatic. Divalent and organic mercury can then enter and pollute biological systems. In last instances, Amerindian populations are contaminated after consumption of carnivorous fish. The mercurial contamination of 35 fish species caught in the Courcibo River, free of gold mining, and Leblond River, whose banks are the location of intensive gold mining, in French Guiana was analyzed and a relationship was found with the level of each species among the trophic web. Results showed a mercury amplification all along the food web: the ratio between the extreme muscle mercury concentrations in piscivorous species (14.3 μg/g dry weight, for Acestrorhynchus guianensis) and herbivorous species (0.02 μg/g dw, for Myleus ternetzi) was 715 . The final predators in this food web are human beings, and consequently high mercury levels are always quantified in hair of Amerindian community members . In 1997, 64% of Wayana Amerindians living in the upper marshes of the Maroni River presented a hair mercury concentration exceeding the safety limit set up by the World Health Organization, and above which adverse effects on brain development are likely to occur (10 μg/g or 10 ppm) . In 2005, this proportion reached 84%, indicating that the problem of mercury contamination increases with time. All individuals one year and older were ingesting, through fish consumption, a mercury dose more important than the security limit set to 200 μg/week. Four carnivorous fish species, Pseudoplatystoma fasciatum, Hoplias aimara, Ageneiosus brevifilis, and Serrasalmus rhombeus, represented at least 72% of the total mercury ingested by the Wayana families . A survey has been carried out in French Guiana showing a significant correlation between mercury contamination levels and neurological impairments. Amerindian children from the upper Maroni were highly contaminated with a mean of 12 ppm in hair, and were afflicted by neurological disorders such as poorer coordination of the legs, and decreased performance in the Stanford-Binet copying score . Taking this correlation into consideration, our long-term goal is to ascertain whether the mercury found in the fish consumed by the Wayana Amerindians is the source of the observed troubles, and if so whether this mercurial intoxication observed among the Amerindian populations is likely to endanger their lives.
To achieve this objective, we chose the rodent model (mouse). The idea was to mimic as closely as possible the Wayanas' contamination mode. Therefore, we decided to incorporate lyophilized fish flesh into the preparation of mice alimentary pellets. This flesh originated from fish contaminated by mercury in their natural habitat and caught in the Sinnamary River in French Guiana. More precisely, the Hoplias aimara species, which Amerindians are fond of, was chosen because this fish is highly contaminated by methylmercury (4 to 12 μg/g dw), and because this single species represents 27% of the Wayanas' dietary mercury intake and 10.7% of the total flesh they consume . A more classical approach consisting in dispersing a given quantity of methylmercury within diet preparations was precluded because the supramolecular form under which methylmercury enters the body is of crucial importance. Indeed it has been shown that methylmercury contained in fish flesh was mainly associated to proteinaceous aliphatic thiols . Therefore, one can suspect a different trophic transfer rate through the intestinal barrier, and a different early toxicity for ingested free and protein-bound methylmercury. In line with this, a higher faecal excretion and lower tissue accumulation, as well as metallothionein induction in rats following exposure to methylmercury naturally incorporated in fish compared to methylmercury chloride added to the same matrix have been reported .
Dietary MeHg is readily and efficiently absorbed by the human gastrointestinal tract, to a reported level of 95% to 100% . However, nothing is known about the MeHg trophic transfer rate in mice at such low exposure doses. Thus, to establish our model, and although our long-term objective was a follow-up of the dietary MeHg impact on mice metabolism all along the animals' life, we first had to determine the fish regimen that given to mice would as closely as possible mimic a human contamination. More precisely, the main goal of this experiment was to select the MeHg-contaminated diet leading to kidney and brain Hg concentrations in the range of what has been recorded in human kidneys and brains of heavy fish consumers in a general population [11, 12].
Three fish flesh-containing diets were made up from a basic vegetarian diet. These diets incorporated 0.1, 1, and 7.5% lyophilized H. aimara flesh, yielding mercury concentrations of 5.4, 62, and 520 ng per g of food pellets, respectively. After feeding mice one month with such regimens, the effects of mercury-containing fish flesh as compared to the control diet were assessed through tissue mercury content analysis, gene expression quantification, muscle mitochondrial respiration assays, and tests for anxiety.
Preparation of the mice diets
The composition of the diets used.a
Diet (percentage of fish flesh in food)
Saturated fatty acids
Monounsaturated fatty acids
C14:1 (w5) Myristoleic
C16:1 (w7) Palmitoleic
C18:1 (w9) Oleic
Polyunsaturated fatty acids
C18:2 (w6) Linoleic
C18:3 (w3) Linolenic
C20:4 (w6) Arachidonic
C22:6 (w3) Cervonic (DHA)
The metal composition of the diets used. a
Diet (percentage of fish flesh in food)
Mice treatment and tissue sampling
Subjects were naïve male mice of the C57Bl/6 Jico inbred strain obtained from IFFA Credo (Lyon, France) at the age of 3 weeks weighing 8.2 ± 0.1 g. They were socially housed in standard conditions: room temperature (23°C), 12/12 light cycles and ad libitum food and water. Experiments were performed in compliance with the European Community Council directive of 24 November 1986 (8616091 EEC). Four groups of 8 mice were fed for one month as follows: one with the control RM1 diet, and the three other ones with 0.1, 1, and 7.5% fish flesh supplemented RM1 diets. At the end of the exposure period, mice were subjected to a cross maze test, in order to quantify anxiety. Thereafter, mice were killed by decapitation, blood was immediately collected, and all the tissues were dissected for mercury quantification and gene expression analysis. For muscle fiber bioenergetics, gastrocnemius, a fast-twitch skeletal muscle was dissected and immediately placed in a cooled solution of buffer A containing 2.8 mM CaK2EGTA, 7.2 mM K2EGTA, 6.5 mM MgCl2, 5.7 mM Na2ATP, 15 mM phosphocreatine, 0.5 mM dithiothreitol, 50 mM potassium methanesulfonate, 20 mM imidazole, and 20 mM taurine (pH 7.1).
Anxiety test using a cross maze
This test is one of the most widely used tests for assessing anxiety states of individuals [14, 15]. The cross maze was elevated to 50 cm above the floor and consisted of two open and two closed arms (fenced on three sides). In such a maze, mice experience open bright spaces as worrying and closed dark ones as reassuring. Thus, open arms of the maze and especially their extremities will be experienced by the animals as deeply anxiety-producing places, centre as mildly anxiety-producing whereas closed arms will be felt as comforting places. Individuals were tested in the maze for 5 min as previously described . Animals were placed in the centre of the maze with the nose pointing toward a closed arm; measures reflecting the anxiety state were measured, as follows: the time spent in the open arms, centre, and closed arms of the maze (data presented as percentage ratios of the time respectively spent in these zones to the total test time), the number of excursions into the open and closed arms, also expressed as percentage ratios, the time spent at the extremity of open and closed arms expressed in seconds, the total number of entries and exits from arms mostly reflecting the general activity of the mice. The maze was thoroughly cleaned and dried with clean tissues after each individual had been tested. All experiments were performed under normal laboratory illumination (1 × 100 W white light) during light phase of the light-dark cycle.
Statistical analysis of anxiety state parameters was performed with a non-parametric Kruskall-Wallis analysis of variance method followed by a Mann-Whitney U test.
Total Hg concentrations in mice tissues were determined by flameless atomic absorption spectrometry. Analyses were carried out automatically after thermal decomposition at 750°C under an oxygen flow (AMA 254, Prague, Czech Republic). The detection limit was 0.01 ng Hg. The validity of the analytical methods was checked during each series of measurements using three standard biological reference materials (TORT2, DOLT2 and DOLT3); Hg values were consistently within the certified value range (data not shown). Stomach and intestines were washed from processed food and faecal matter before analysis.
Gene expression analysis
Specific primer pairs for the Mus musculus genes used. a
Interindividual variability for each experimental condition was defined by mean ± standard deviation (n = 3). Significant differential gene expression levels between control mice and fish-fed mice in the four organs were determined using the nonparametric Mann-Whitney U-test (p < 0.05).
Mitochondrial respiration measurements on skinned muscle fibers
Gastrocnemius and quadriceps muscular fibers (between 10 and 20 mg) collected on the posterior limbs of mice were permeabilized with saponin, a natural smooth detergent, in order to make mitochondria accessible to respiratory substrates added in the media. Bundles of fibers were incubated for 20 min in 5 ml of solution A containing saponin 50 μg/ml as described . The bundles were then washed twice for 15 min in solution B (EGTA 10 mM, Mg2+ 3 mM, taurine 20 mM, dithiotreitol 0.5 mM, imidazole 20 mM, K+MES 0.1 M pH 7.0, phosphate 3 mM and 5 mg/ml fatty-acid-free bovine serum albumin) to remove saponin. All procedures were carried out at 4°C with extensive stirring. Finally, the preparations remained stable in the ice-cold solution B for 3 h. Mitochondrial oxygen consumption was monitored at 30°C in a 1 ml thermostatically controlled chamber equipped with a Clark oxygen electrode connected to a computer that gives an on-line display of the respiratory rate value (Hansatech, OXY1 system). The oxygraph cuvette contained one bundle of permeabilized muscles (around 12 mg) in 1 ml of solution B with Ap5A (di(Adenosine-5') pentaphosphate) 50 μM, iodoacetate 10 mM, EDTA 0.2 mM and the respiratory substrates (pyruvate 10 mM in the presence of malate 10 mM). State 3 was obtained by addition of 2 mM ADP. After each respiration measurement, the bundle of fibers was removed from the cuvette, dried and weighed to allow expression of the respiratory rates in ng atom O/min/mg of fibers. The respiratory control ratio (RCR) is defined as the ratio of state 3 (in the presence of ADP) to state 4 (in absence of ADP) respiratory rates.
Cytochrome c oxidase activity
Cytochrome c oxidase activity was monitored by inhibiting the upstream components of the respiratory chain with rotenone and antimycin, and by using 3 mM ascorbate and 0.5 mM TMPD as an electron donor system. The respiratory rate was monitored using the polarographic method described above .
Mercury bioaccumulation within mice organs
Mercury bioaccumulation in various tissues. a
Tissue (n = 3)
Diet (Hg dose in food expressed in ng/g)
1 ± 0.8
81 ± 47
1579 ± 165
10364 ± 3103
0.3 ± 0.1
116 ± 20
1435 ± 112
7730 ± 59
0.2 ± 0.04
6.7 ± 1.2
150 ± 14
1135 ± 324
0.2 ± 0.2
8 ± 1
88 ± 2
543 ± 65
5.9 ± 1
64 ± 5
417 ± 39
5 ± 1
63 ± 3
299 ± 31
2.5 ± 1.5
77 ± 18
1111 ± 232
2.1 ± 0.9
70 ± 4
614 ± 90
1.8 ± 0.7
64 ± 12
576 ± 69
1.5 ± 0.3
54 ± 4
517 ± 137
1.2 ± 0.3
45 ± 7
317 ± 119
1.2 ± 0.4
15.3 ± 3
201 ± 28
1.1 ± 0.6
34 ± 4
298 ± 36
Impact of fish-containing diets on gene expression
Comparative basal gene expressions in various tissues. a
65.103 ± 11.103
2048 ± 1354
8192 ± 1214
104.104 ± 84.104
8 ± 6.8
256 ± 104
128 ± 8.5
2048 ± 279
4 ± 1.9
2 ± 1.4
16 ± 2.2
8 ± 4.3
8 ± 2.5
4 ± 1.3
4 ± 0.9
4 ± 3.8
1024 ± 430
64 ± 3
64 ± 33
512 ± 482
32 ± 27
512 ± 4.9
32 ± 4.4
256 ± 51
8 ± 3.2
16 ± 2.1
16 ± 8.8
512 ± 189
4 ± 3.7
0.25 ± 0.01
0.5 ± 0.4
16 ± 9.5
Differential gene expressions in various tissues. a
Food (ng Hg/g)
In summary, a net genetic response could be observed for mercury concentrations accumulated within tissues as weak as 0.15 ppm in the liver, 1.4 ppm in the kidneys, and 0.4 ppm in the hippocampus.
Impact of fish-containing diets on muscle mitochondrial respiration
Respiratory rates assayed on skinned muscle fibers. a
Diet (Hg dose in food expressed in ng/g)
Oxygen consumption (ng atom O/min/mg fw)
State 4 of respiration
1.6 ± 0.5
1.4 ± 0.2
1.3 ± 0.2
1.8 ± 0.4
State 3 of respiration
2.9 ± 0.7
*1.6 ± 0.3
*1.8 ± 0.4
2.1 ± 0.5
2.0 ± 0.2
*1.1 ± 0.2
*1.3 ± 0.2
*1.17 ± 0.05
4.2 ± 0.8
*1.9 ± 0.3
3.2 ± 0.5
3.3 ± 0.3
Impact of fish-containing diets on anxiety level
Behavior of mice fed with mercury-containing diets in the cross maze test. a
Diet (Hg dose in food expressed in ng/g)
Control diet (n = 6)
5.4 (n = 8)
62 (n = 8)
520 (n = 8)
Number of entries into open arms, %
28 ± 3.5
**13.7 ± 4.2
*21 ± 2.6
26.3 ± 3
Time spent in open arms, %
20.4 ± 2.9
*7.7 ± 2.9
*8.7 ± 1.6
14.7 ± 3.8
Time spent at the extremity of open arms, (sec)
30.9 ± 8.7
*13.4 ± 6.8
*14.6 ± 3.4
23.6 ± 8.7
Number of entries into closed arms, %
71.5 ± 4.2
**86.3 ± 4.2
*79 ± 2.6
73.7 ± 3
Time spent in closed arms, %
41.4 ± 2.8
**61.5 ± 4.3
**61 ± 2.6
49.4 ± 3.9
Time spent at the extremity of closed arms, (sec)
112.4 ± 13.6
**159.1 ± 14.2
**158.3 ± 8.5
116.5 ± 11.2
Time spent in center, %
38.3 ± 2
*31 ± 2.2
**30.2 ± 1.7
36.3 ± 1.7
Surprisingly, in view of the results obtained with lighter diets, mice fed with the 7.5% fish-containing diet did not exhibit any statistically relevant differences in anxiety-like state behavior compared to controls.
Altogether, our results show that a vegetarian diet containing as little as 0.1% of mercury-contaminated fish is able to trigger, after only one month of exposure, bioenergetical disorders in skeletal muscles, a genetic response in liver and kidneys, and an increase in the anxiety-driven behavior of mice demonstrating that the aimara flesh is harmful. Although we cannot rule out that one or several toxic compounds, other than mercury, are present in the aimara flesh and act additionally to or synergistically with methylmercury, we now have solid arguments to incriminate methylmercury as the toxic compound being delivered by the fish flesh-containing diets to mice.
First, methylmercury is the only known toxic compound contaminating the food web of the Sinnamary River, and apart from clandestine gold mining activities, no sources of organic xenobiotics have been recorded so far in this part of the Amazonian jungle.
Second, the mercury accumulation in mice tissues is dependent on the diet fish content.
Third, gene expression studies are now powerful enough to discriminate and classify toxicants on the basis of unique gene expression profiles induced by putative toxic actions. Recently, this concept has been applied using DNA microarrays to evaluate the putative toxicity of environmental pollutants, yielding some chemical-specific gene expression patterns in mice tissues and cultured cells [18–21]. This concept also applies to metal intoxication: human lung cells have been exposed to cadmium chloride, sodium dichromate, nickel subsulfide or sodium arsenite. Using a 1200-gene microarray, it was shown that only three to seven genes overlapped among any two metal treatments . In our hands, using a panel of selected genes, we could make a comparison of zebrafish genes differentially expressed in direct cadmium and trophic methylmercury contamination conditions. p53 and sod1 genes were specific to methylmercury whereas hsp70, mt1, and pyc were specific to cadmium. Among other genes, bax, coxI, sod2, and mt2 were common to both toxicants [23, 24]. Worth noting, the same set of genes as those activated by methylmercury in zebrafish was also responding in mice fed with fish-containing diets. Indeed, this treatment stimulated the increased expression of Cox I, Gsta4, Mt2, Sod2, and Sod3 genes in liver, Cox I, Bax, Fos, and Sod3 genes in kidneys, and that of Bax, Fos, Mt1, Mt2, Sod2, and Sod3 genes in hippocampus. This pattern of gene expression was an expected response in the case of a mercurial contamination, and is unlikely to be caused by other toxic compounds. Mt gene overexpression is a hallmark of divalent cadmium and mercury exposure and has been observed in several animal tissues and cultured cells in addition to our zebrafish study: for instance in lungs of rats having inhaled mercury vapor , and in human hepatoma cells treated with cadmium or mercuric chloride . In contrast, DNA microarray analysis showed that: 1/whereas cadmium chloride indeed triggered overexpression of Mt1 and Mt2 genes in mice liver, benzopyrene and trichloroethylene were unable to do so whatever the tested doses ; 2/whereas cadmium chloride and mercury chloride up-regulated MT1 gene in human hepatoma HepG2 cells, 2,3-dimethoxy-1,4-naphtoquinone exerted no effects, and phenol and N-nitrosodimethylamine down-regulated this gene ; 3/in rat liver, phenobarbital, gemfibrozil and clofibrate could not induce up-regulation of any of the genes we found stimulated in mice liver, with the exception of Gst gene in the case of phenobarbital. In fact, gemfibrozil and clofibrate rather down-regulated Mt1 and Mt2 genes ; 4/the same holds true in human hepatoma cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin, in which the only gene differentially expressed among those up-regulated in our mice liver was Cox I. However, it was 2.4-times down-regulated in this cell type . Consistent with our findings, rats fed with mercury-contaminated rice produced near the Wanshan mercury mine in China were subjected to an oxidative stress materialized by an 87% increase in free radicals, a modification of the activity of superoxide dismutase, and the differential up-regulation of Fos gene in the hippocampus and cortex [26, 27].
Fourth, our results on muscle mitochondrial respiration are fully concordant with the long-known effects of methylmercury on mitochondria of human and rat liver, i.e. state 3 respiration was inhibited by 10 to 50 nmol of methylmercury per mg of mitochondrial protein, and the resulting loss in membrane potential was the major cause of uncoupling . In addition, purified beef heart cytochrome c oxidase is up to 50% inactivated by mercury chloride and ethylmercury , and in rats given methylmercury orally at a concentration of 5 μg/g per day for 12 days, mitochondria of skeletal muscles were affected by a decrease in cytochrome c oxidase and succinate dehydrogenase activities . Methylmercury treatment also resulted in impaired mitochondrial dehydrogenase activity in cultured rat cerebellar granule cells  and mouse cerebellar neurons and astrocytes . This is in keeping with our study on zebrafish fed with methylmercury-contaminated diet. After 49 days of contamination, state 3 mitochondrial respiration was reduced by 80%, and the cytochrome c oxidase activity reduced by 60% in saponin-permeabilized muscle fibers .
The biggest impact of fish diets on behavior, mitochondrial respiratory rates and kidney genes expression was observed with the low and mid level diets, not with the high level one. Although surprising at first glance, many experimental results are now showing that above a given dose of contaminant, the effects observed at low dose vanish or differ qualitatively. For instance, the effects of arsenic at 5 or 50 μM on human lung cells exposed for 4 hours were compared. Increasing the dose of arsenic from 5 to 50 μM did not simply increase the magnitude of the change in the same set of genes or induce additional genes response. Rather, a completely different pattern of gene response between the lower and the higher dose was observed. Over the 1200 genes examined at both doses, only 16 of the 160 affected genes were altered at both doses . In another study authors carried out a serial analysis of gene expression (SAGE) in kidneys from mice exposed to chronic or acute uranyl nitrate contamination [35, 36]. Only 16 genes were common to both SAGE lists and expressed the same way; 147 genes were differentially regulated in either one of the two conditions; 10 genes were common to both SAGE lists but expressed the opposite way, i.e. up-regulated under chronic exposure but down-regulated under acute exposure. These comparative patterns of gene expression data indicate that when shifting from chronic to acute exposure the intensity of gene response does not increase as might have been expected but rather that the qualitative nature of the gene response is completely changed resulting in a modified tissue metabolism. In keeping with this, it has been shown that: a/uranium is an endocrine disrupter in mice at low but not at high doses ; b/after 7 days of exposure, copper induced in the aquatic plant Hydrilla verticillata an increase of superoxide dismutase, glutathione peroxidase and catalase activities at low but not at high doses ; c/cadmium triggered greater genotoxic damages on Xenopus laevis larvae at 0.5 than at 1 mg/l ; d/carbon nanotubes induced in rainbow trout gills and intestine an increase of the Na+K+-ATPase activity at low but not at high doses ; e/nanofullerenes induced in largemouth bass gills and liver a change in the pattern of lipid peroxidation at 0.5 ppm but not at 1 ppm . These results suggest a model in which low doses of pollutant cause mild effects compatible with life, allowing animal resistance-through adaptive response, whereas higher doses trigger acute effects threatening animal's life, resulting in general stress instead of adaptive response. Additionally, the highest regimen is rich in fish flesh and it has been argued that the beneficial influence of nutrients from fish may counter any adverse effects of MeHg on the developing nervous system .
Toxic effects of mercury on various cell lines.
Organisms and species
Bacteria Escherichia coli
Strains devoid of R plasmids
Growth inhibition: MIC = 11.5 μM HgCl2.
Yeast Saccharomyces cerevisiae
Growth inhibition: MIC = 2 μM MeHgCl at 24 h.
Clam Mya arenaria
Phagocytosis inhibition: IC50 = 0.44 μM MeHgCl at 18 h.
Earthworm Lumbricus terrestris
Phagocytosis inhibition: IC50 = 0.1 μM MeHgCl and 0.5 μM HgCl2 at 18 h.
Mosquito Aedes albopictus
Cell line C6/36
Cell viability: for serum deprived cultures, LD50 = 2.1 μM MeHgCl and 2.5 μM HgCl2 at 24 h; for cultures with fetal calf serum, LD50 = 5.5 μM MeHgCl and 12 μM HgCl2 at 24 h.
Cell growth inhibition: IC50 = 1 μM MeHgCl and 18.4 μM HgCl2 at 18 days.
Cell viability: 49% cell death with 10 μM MeHgCl at 6 h.
Fish fathead minnow
Cell viability: EC50 = 1.55 μM MeHgOH at 96 h.
Renal proximal tubule cells
Cell viability: LC50 = 6.1 μM MeHgCl and 34.2 μM HgCl2 at 24 h.
Cerebellar granule cells
50% apoptotic cells with 1 μM MeHgCl for 9 h; 30% apoptotic cells and 60% reduction in mitochondrial dehydrogenases with 2.5 μM MeHgCl for 1 h.
Embryonic neural stem cells
90% cell death with 0.5 μM MeHgCl for 24 h; 37% apoptotic cells with 0.1 μM MeHgCl for 24 h.
Cytolethality: 8 μM MeHgCl for 24 h.
50% reduction in mitochondrial activity with 5 μM MeHgCl for 1 h.
40% reduction in mitochondrial activity with 5 μM MeHgCl for 1 h.
Cell death: 20 μM MeHgCl for a few days.
Necrotic cell death: 15 μM MeHgCl for 13 min.
Multipotent neural stem cell line C17.2
45% cell death with 2 μM MeHgCl at 24 h; 20% apoptotic cells with 0.5 μM at 24 h.
YAC-1 murine Moloney virus transformed lymphoma cell line
50% cell death with 25 μM MeHgCl at 4 h.
Cell viability: 8 μM MeHgCl at 24 h.
Cell viability: 8 μM MeHgCl at 4 h.
Cell viability: LC50 = 6.5 μM MeHgCl at 24 h.
Cell viability: LC50 = 8.1 μM MeHgCl at 24 h.
Cell viability: LC50 = 6.9 μM MeHgCl at 24 h.
The 7.5% fish-containing diet resulted in Hg brain concentrations equivalent to acute exposure cases and therefore cannot be useful to mimic human environmental cases. The 1% fish-containing diet yielded after only one month exposure, blood, kidney, and brain Hg concentrations in the range of what has been recorded in human blood, kidneys, and brains of heavy fish consumers in a general population. The 0.1% fish-containing diet brings to mice the same mercury contamination pressure as that afflicting the Wayana Amerindians assuming a Hg trophic transfer rate of 100%, and it can be expected that after several months, the mercury levels in mice tissues be equivalent to those observed after one month of feeding with diet containing 1% fish flesh. Since the 0.1% fish-containing regimen proved to affect gene expression, muscle mitochondrial respiration, and triggered an anxiety-driven behavior in mice, our study will be pursued with such a regimen for an extended time length encompassing the mouse lifespan in order to get a precise panorama of the impact of mercury-contaminated fish consumption all along the animals' life.
complementary desoxyribonucleic acid
ethylene glycol tetraacetic acid
polymerase chain reaction
respiratory control ratio
This work was supported by the French National Research Agency, program "Santé-environnement et santé-travail", and by a grant from University Pierre and Marie Curie Paris VI for international projects.
- Harada M: Neurotoxicity of methylmercury: Minamata and the Amazon. Mineral and Metal Neurotoxicology. Edited by: Yasui M, Strong MJ, Ota K, Verity MA. 1997, London: CRC Press, 177-188.Google Scholar
- Grandjean P, Weiche P, White RF, Debes F, Araki S, Yokoyama K, Murata K, Sorensen N, Dahl R, Jorgensen PJ: Cognitive deficit in 7-year-old children with prenatal exposure to methylmercury. Neurotoxicol Teratol. 1997, 19: 417-428. 10.1016/S0892-0362(97)00097-4.View ArticleGoogle Scholar
- Durrieu G, Maury-Brachet R, Boudou A: Goldmining and mercury contamination of the piscivorous fish Hoplias aimara in French Guiana (Amazon basin). Ecotoxicol Environ Saf. 2005, 60: 315-323. 10.1016/j.ecoenv.2004.05.004.View ArticleGoogle Scholar
- Cordier S, Grasmick C, Paquier-Passelaigue M, Mandereau L, Weber JP, Jouan M: Mercury exposure in French Guiana: levels and determinants. Arch Environ Health. 1998, 53: 299-303.View ArticleGoogle Scholar
- World Health Organization. International Programme on Chemical Safety: Methylmercury. Environmental Health Criteria 101. Geneva. 1990, [http://www.inchem.org/documents/ehc/ehc/ehc101.htm]Google Scholar
- Fréry N, Maury-Brachet R, Maillot E, Deheeger M, de Merona B, Boudou A: Gold-mining activities and mercury contamination of native amerindian communities in French Guiana: key role of fish in dietary uptake. Environ Health Perspect. 2001, 109: 449-456. 10.2307/3454702.View ArticleGoogle Scholar
- Cordier S, Garel M, Mandereau L, Morcel H, Doineau P, Gosme-Seguret S, Josse D, White R, Amiel-Tison C: Neurodevelopmental investigations among methylmercury-exposed children in French Guiana. Environ Res. 2002, 89: 1-11. 10.1006/enrs.2002.4349.View ArticleGoogle Scholar
- Harris HH, Pickering IJ, George GN: The chemical form of mercury in fish. Science. 2003, 301: 1203-10.1126/science.1085941.View ArticleGoogle Scholar
- Berntssen MH, Hylland K, Lundebye AK, Julshamn K: Higher faecal excretion and lower tissue accumulation of mercury in Wistar rats from contaminated fish than from methylmercury chloride added to fish. Food Chem Toxicol. 2004, 42: 1359-1366. 10.1016/j.fct.2004.03.013.View ArticleGoogle Scholar
- Canuel R, de Grosbois SB, Lucotte M, Atikessé L, Larose C, Rheault I: New evidence on the effects of tea on mercury metabolism in humans. Arch Environ Occup Health. 2006, 61: 232-238. 10.3200/AEOH.61.5.232-238.View ArticleGoogle Scholar
- Barregård L, Svalander C, Schütz A, Westberg G, Sällsten G, Blohmé I, Mölne J, Attman PO, Haglind P: Cadmium, mercury, and lead in kidney cortex of the general Swedish population: a study of biopsies from living kidney donors. Environ Health Perspect. 1999, 107: 867-871. 10.2307/3454473.View ArticleGoogle Scholar
- Björkman L, Lundekvam BF, Laegreid T, Bertelsen BI, Morild I, Lilleng P, Lind B, Palm B, Vahter M: Mercury in human brain, blood, muscle and toenails in relation to exposure: an autopsy study. Environ Health. 2007, 6: 30-10.1186/1476-069X-6-30.View ArticleGoogle Scholar
- Boudou A, Maury-Brachet R, Coquery M, Durrieu G, Cossa D: Synergic effect of gold mining and damming on mercury contamination in fish. Environ Sci Technol. 2005, 39: 2448-2454. 10.1021/es049149r.View ArticleGoogle Scholar
- Lister RG: The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology (Berl). 1987, 92 (2): 180-185. 10.1007/BF00177912.View ArticleGoogle Scholar
- Rodgers RJ, Cole JC: The elevated plus-maze: pharmacology, methodology and ethology. Ethology and Psychopharmacology. Edited by: Cooper SJ, Hendrie CA. 1994, Chichester, New-York: John Wiley & Sons Ltd, 85-109.Google Scholar
- Dubrovina NI, Tomilenko RA: Characteristics of extinction of a conditioned passive avoidance reflex in mice with different levels of anxiety. Neurosci Behav Physiol. 2007, 37: 27-32. 10.1007/s11055-007-0145-x.View ArticleGoogle Scholar
- Letellier T, Malgat M, Coquet M, Moretto B, Parrot-Roulaud F, Mazat JP: Mitochondrial myopathy studies on permeabilized muscle fibers. Pediatr Res. 1992, 32: 17-22. 10.1203/00006450-199207000-00004.View ArticleGoogle Scholar
- Bartosiewicz M, Penn S, Buckpitt A: Applications of gene arrays in environmental toxicology: fingerprints of gene regulation associated with cadmium chloride, benzo(a)pyrene, and trichloroethylene. Environ Health Perspect. 2001, 109: 71-74. 10.2307/3434924.View ArticleGoogle Scholar
- Hamadeh HK, Bushel PR, Jayadev S, Martin K, DiSorbo O, Sieber S, Bennett L, Tennant R, Stoll R, Barrett JC, Blanchard K, Paules RS, Afshari CA: Gene expression analysis reveals chemical-specific profiles. Toxicol Sci. 2002, 67: 219-231. 10.1093/toxsci/67.2.219.View ArticleGoogle Scholar
- Kawata K, Yokoo H, Shimazaki R, Okabe S: Classification of heavy-metal toxicity by human DNA microarray analysis. Environ Sci Technol. 2007, 41: 3769-3774. 10.1021/es062717d.View ArticleGoogle Scholar
- Puga A, Maier A, Medvedovic M: The transcriptional signature of dioxin in human hepatoma HepG2 cells. Biochem Pharmacol. 2000, 60: 1129-1142. 10.1016/S0006-2952(00)00403-2.View ArticleGoogle Scholar
- Andrew AS, Warren AJ, Barchowsky A, Temple KA, Klei L, Soucy NV, O'Hara KA, Hamilton JW: Genomic and proteomic profiling of responses to toxic metals in human lung cells. Environ Health Perspect. 2003, 111: 825-838.Google Scholar
- Gonzalez P, Dominique Y, Massabuau JC, Boudou A, Bourdineaud JP: Comparative effects of dietary methylmercury on gene expression in liver, skeletal muscle, and brain of the zebrafish (Danio rerio). Environ Sci Technol. 2005, 39: 3972-3980. 10.1021/es0483490.View ArticleGoogle Scholar
- Gonzalez P, Baudrimont M, Boudou A, Bourdineaud JP: Comparative effects of direct cadmium contamination on gene expression in gills, liver, skeletal muscles and brain of the zebrafish (Danio rerio). Biometals. 2006, 19: 225-235. 10.1007/s10534-005-5670-x.View ArticleGoogle Scholar
- Liu J, Lei D, Waalkes MP, Beliles RP, Morgan DL: Genomic analysis of the rat lung following elemental mercury vapor exposure. Toxicol Sci. 2003, 74: 174-181. 10.1093/toxsci/kfg091.View ArticleGoogle Scholar
- Jie XL, Jin GW, Cheng JP, Wang WH, Lu J, Qu LY: Consumption of mercury contaminated rice induces oxidative stress and free radical aggravation in rats. Biomed Environ Sci. 2007, 20: 84-89.Google Scholar
- Cheng JP, Hu WX, Liu XJ, Zheng M, Shi W, Wang WH: Expression of c-fos and oxidative stress on brain of rats reared on food from mercury-selenium coexisting mining area. J Environ Sci (China). 2006, 18: 788-792.Google Scholar
- Sone N, Larsstuvold MK, Kagawa Y: Effect of methyl mercury on phosphorylation, transport, and oxidation in mammalian mitochondria. J Biochem. 1977, 82: 859-868.Google Scholar
- Mann AJ, Auer HE: Partial inactivation of cytochrome c oxidase by nonpolar mercurial reagents. J Biol Chem. 1980, 255: 454-458.Google Scholar
- Usuki F, Yasutake A, Matsumoto M, Umehara F, Higuchi I: The effect of methylmercury on skeletal muscle in the rat: a histopathological study. Toxicol Lett. 1998, 94: 227-232. 10.1016/S0378-4274(98)00022-8.View ArticleGoogle Scholar
- Castoldi AF, Barni S, Turin I, Gandini C, Manzo L: Early acute necrosis, delayed apoptosis and cytoskeletal breakdown in cultured cerebellar granule neurons exposed to methylmercury. J Neurosci Res. 2000, 59: 775-787. 10.1002/(SICI)1097-4547(20000315)59:6<775::AID-JNR10>3.0.CO;2-T.View ArticleGoogle Scholar
- Kaur P, Aschner M, Syversen T: Glutathione modulation influences methyl mercury induced neurotoxicity in primary cell cultures of neurons and astrocytes. Neurotoxicology. 2006, 27: 492-500. 10.1016/j.neuro.2006.01.010.View ArticleGoogle Scholar
- Cambier S, Bénard G, Mesmer-Dudons N, Gonzalez P, Rossignol R, Brèthes D, Bourdineaud JP: At environmental relevant low dose, dietary methylmercury inhibits the mitochondrial electron transfer chain and the production of ATP in skeletal muscles of the zebra fish (Danio rerio). Abstract Book of the 17th annual meeting of the Society of Environmental Toxicology and Chemistry: 20–24 May 2007; Porto, Portugal. 2007, SETAC EuropeGoogle Scholar
- Andrew AS, Warren AJ, Barchowsky A, Temple KA, Klei L, Soucy NV, O'Hara KA, Hamilton JW: Genomic and proteomic profiling of responses to toxic metals in human lung cells. Environ Health Perspect. 2003, 111: 825-835.Google Scholar
- Taulan M, Paquet F, Maubert C, Delissen O, Demaille J, Romey MC: Renal toxicogenomic response to chronic uranyl nitrate insult in mice. Environ Health Perspect. 2004, 112: 1628-1635.View ArticleGoogle Scholar
- Taulan M, Paquet F, Argiles A, Demaille J, Romey MC: Comprehensive analysis of the renal transcriptional response to acute uranyl nitrate exposure. BMC Genomics. 2006, 7: 2-10.1186/1471-2164-7-2.View ArticleGoogle Scholar
- Raymond-Whish S, Mayer LP, O'Neal T, Martinez A, Sellers MA, Christian PJ, Marion SL, Begay C, Propper CR, Hoyer PB, Dyer CA: Drinking water with uranium below the U.S. EPA water standard causes estrogen receptor-dependent responses in female mice. Environ Health Perspect. 2007, 115: 1711-1716.View ArticleGoogle Scholar
- Srivastava S, Mishra S, Tripathi RD, Dwivedi S, Gupta DK: Copper-induced oxidative stress and responses of antioxidants and phytochelatins in Hydrilla verticillata (L.f.) Royle. Aquat Toxicol. 2006, 80: 405-415. 10.1016/j.aquatox.2006.10.006.View ArticleGoogle Scholar
- Mouchet F, Gauthier L, Baudrimont M, Gonzalez P, Mailhes C, Ferrier V, Devaux A: Comparative evaluation of the toxicity and genotoxicity of cadmium in amphibian larvae (Xenopus laevis and Pleurodeles waltl) using the comet assay and the micronucleus test. Environ Toxicol. 2007, 22: 422-435. 10.1002/tox.20267.View ArticleGoogle Scholar
- Smith CJ, Shaw BJ, Handy RD: Toxicity of single walled carbon nanotubes to rainbow trout, (Oncorhynchus mykiss): respiratory toxicity, organ pathologies, and other physiological effects. Aquat Toxicol. 2007, 82: 94-109. 10.1016/j.aquatox.2007.02.003.View ArticleGoogle Scholar
- Oberdörster E: Manufactured nanomaterials (fullerenes, C60) induce oxidative stress in the brain of juvenile largemouth bass. Environ Health Perspect. 2004, 112: 1058-1062.View ArticleGoogle Scholar
- Myers GJ, Davidson PW, Cox C, Shamlye CF, Palumbo D, Cernichiari E, Sloane-Reeves J, Wildning GE, Kost J, Huang LS, Clarkson TW: Prenatal methylmercury exposure from ocean fish consumption in the Seychelles child development study. Lancet. 2003, 361: 1686-1692. 10.1016/S0140-6736(03)13371-5.View ArticleGoogle Scholar
- Ekino S, Susa M, Ninomiya T, Imamura K, Kitamura T: Minamata disease revisited: an update on the acute and chronic manifestations of methyl mercury poisoning. J Neurol Sci. 2007, 262: 131-144. 10.1016/j.jns.2007.06.036.View ArticleGoogle Scholar
- Lemire M, Mergler D, Fillion M, Passos CJ, Guimarães JR, Davidson R, Lucotte M: Elevated blood selenium levels in the Brazilian Amazon. Sci Total Environ. 2006, 366: 101-111. 10.1016/j.scitotenv.2005.08.057.View ArticleGoogle Scholar
- Nakahara H, Ishikawa T, Sarai Y, Kondo I, Kozukue H: Mercury resistance and R plasmids in Escherichia coli isolated from clinical lesions in Japan. Antimicrob Agents Chemother. 1977, 11: 999-1003.View ArticleGoogle Scholar
- Naganuma A, Miura N, Kaneko S, Mishina T, Hosoya S, Miyairi S, Furuchi T, Kuge S: GFAT as a target molecule of methylmercury toxicity in Saccharomyces cerevisiae. FASEB J. 2000, 14: 968-972.Google Scholar
- Brousseau P, Pellerin J, Morin Y, Cyr D, Blakley B, Boermans H, Fournier M: Flow cytometry as a tool to monitor the disturbance of phagocytosis in the clam Mya arenaria hemocytes following in vitro exposure to heavy metals. Toxicology. 2000, 142: 145-156. 10.1016/S0300-483X(99)00165-1.View ArticleGoogle Scholar
- Fugère N, Brousseau P, Krzystyniak K, Coderre D, Fournier M: Heavy metal-specific inhibition of phagocytosis and different in vitro sensitivity of heterogeneous coelomocytes from Lumbricus terrestris (Oligochaeta). Toxicology. 1996, 109: 157-166. 10.1016/0300-483X(96)03315-X.View ArticleGoogle Scholar
- Braeckman B, Raes H, van Hoye D: Heavy-metal toxicity in an insect cell line. Effects of cadmium chloride, mercuric chloride and methylmercuric chloride on cell viability and proliferation in Aedes albopictus cells. Cell Biol Toxicol. 1997, 13: 389-397. 10.1023/A:1007425925726.View ArticleGoogle Scholar
- Herculano AM, Crespo-Lopez ME, Lima SM, Picanco-Diniz DL, Do Nascimento JL: Methylmercury intoxication activates nitric oxide synthase in chick retinal cell culture. Braz J Med Biol Res. 2006, 39: 415-418. 10.1590/S0100-879X2006000300013.View ArticleGoogle Scholar
- Devlin EW, Clary B: In vitro toxicity of methyl mercury to fathead minnow cells. Bull Environ Contam Toxicol. 1998, 61: 527-533. 10.1007/s001289900794.View ArticleGoogle Scholar
- Aleo MD, Taub ML, Kostyniak PJ: Primary cultures of rabbit renal proximal tubule cells. III. Comparative cytotoxicity of inorganic and organic mercury. Toxicol Appl Pharmacol. 1992, 112: 310-317. 10.1016/0041-008X(92)90201-3.View ArticleGoogle Scholar
- Tamm C, Duckworth J, Hermanson O, Ceccatelli S: High susceptibility of neural stem cells to methylmercury toxicity: effects on cell survival and neuronal differentiation. J Neurochem. 2006, 97: 69-78. 10.1111/j.1471-4159.2006.03718.x.View ArticleGoogle Scholar
- Omara FO, Flipo D, Brochu C, Denizeau F, Brousseau P, Potworowski EF, Fournier M: Lack of suppressive effects of mixtures containing low levels of methylmercury (MeHg), polychlorinated dibenzo-p-dioxins (PCDDS), polychlorinated dibenzofurans (PCDFS), and aroclor biphenyls (PCBS) on mixed lymphocyte reaction, phagocytic, and natural killer cell activities of rat leukocytes in vitro. J Toxicol Environ Health A. 1998, 54: 561-577. 10.1080/009841098158700.View ArticleGoogle Scholar
- Christensen MM, Ellermann-Eriksen S, Rungby J, Mogensen SC: Comparison of the interaction of methyl mercury and mercuric chloride with murine macrophages. Arch Toxicol. 1993, 67: 205-211. 10.1007/BF01973309.View ArticleGoogle Scholar
- Kuo TC, Lin-Shiau SY: Early acute necrosis and delayed apoptosis induced by methyl mercury in murine peritoneal neutrophils. Basic Clin Pharmacol Toxicol. 2004, 94: 274-281.View ArticleGoogle Scholar
- Yole M, Wickstrom M, Blakley B: Cell death and cytotoxic effects in YAC-1 lymphoma cells following exposure to various forms of mercury. Toxicology. 2007, 231: 40-57. 10.1016/j.tox.2006.11.062.View ArticleGoogle Scholar
- Shenker BJ, Berthold P, Decker S, Mayro J, Rooney C, Vitale L, Shapiro IM: Immunotoxic effects of mercuric compounds on human lymphocytes and monocytes. II. Alterations in cell viability. Immunopharmacol Immunotoxicol. 1992, 14: 555-577. 10.3109/08923979209005411.View ArticleGoogle Scholar
- Sanfeliu C, Sebastia J, Ki SU: Methylmercury neurotoxicity in cultures of human neurons, astrocytes, neuroblastoma cells. Neurotoxicology. 2001, 22: 317-327. 10.1016/S0161-813X(01)00015-8.View ArticleGoogle Scholar
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