Specific Pathways of Dietary Methylmercury and Inorganic Mercury

Article pubs.acs.org/est

Specific Pathways of Dietary Methylmercury and Inorganic Mercury Determined by Mercury Speciation and Isotopic Composition in Zebrafish (Danio rerio) Caiyan Feng,† Zoyne Pedrero,*,† Sophie Gentès,‡ Julien Barre,† Marina Renedo,† Emmanuel Tessier,† Sylvain Berail,† Régine Maury-Brachet,‡ Nathalie Mesmer-Dudons,‡ Magalie Baudrimont,‡ Alexia Legeay,‡ Laurence Maurice,§,∥ Patrice Gonzalez,‡ and David Amouroux† †

Laboratoire de Chimie Analytique, Bio-Inorganique et Environnement, Institut des Sciences Analytiques et de Physico-Chimie pour l’Environnement et les Matériaux (IPREM), CNRS-UPPA-UMR-5254, Hélioparc, 2 Avenue du Président Pierre Angot, F-64053 Pau, France ‡ Université de Bordeaux, EPOC, UMR CNRS 5805, F-33120 Arcachon, France § Observatoire Midi-Pyrénées, Laboratoire de Géosciences Environnement Toulouse, Université Paul Sabatier Toulouse III, 14 avenue Edouard Belin, 31400 Toulouse, France ∥ GET, IRD, F-31400 Toulouse, France S Supporting Information *

ABSTRACT: An original approach is proposed to investigate inorganic (iHg) and methylmercury (MeHg) trophic transfer and fate in a model fish, Danio rerio, by combining natural isotopic fractionation and speciation. Animals were exposed to three different dietary conditions: (1) 50 ng Hg g−1, 80% as MeHg; (2) diet enriched in MeHg 10 000 ng Hg g−1, 95% as MeHg, and (3) diet enriched in iHg 10 000 ng Hg g−1, 99% as iHg. Harvesting was carried out after 0, 7, 25, and 62 days. Time-dependent Hg species distribution and isotopic fractionation in fish organs (muscle, brain, liver) and feces, exhibited different patterns, as a consequence of their dissimilar metabolization. The rapid isotopic re-equilibration to the new MeHg-food source reflects its high bioaccumulation rate. Relevant aspects related to Hg excretion are also described. This study confirms Hg isotopic fractionation as a powerful tool to investigate biological processes, although its deconvolution and fully understanding is still a challenge.

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occurrence of MeHg transformation in less toxic species,11−15 but details about such biological processes are unknown. Isotopic signature adds another dimension to speciation analyses. It is a powerful tool for the investigation of metabolic processes, as demonstrated on the investigation of Cu,16−18 Zn,19,20 and Se,21 in different living organisms. Specifically in the case of Hg, it brings complementary information about the origin and the processes in which Hg is involved. The natural Hg stable isotope variation (isotopic fractionation) has been extensively used in the identification of (pollution) sources in environmental studies.22,23 Recently, a clear trend is observed on its application in biological processes in bacteria,24−26 plants,27 fish,28,29 and humans.30−33 Hg stable isotopes can experiment two types of fractionation: mass dependent fractionation (MDF) and mass independent fractionation (MIF). The first one has been demonstrated to be helpful for tracking Hg metabolic processes as species

INTRODUCTION

Mercury (Hg) toxicity can be easily biomagnified in the trophic chain. In aquatic ecosystems, inorganic Hg (iHg) from sediments is transformed in methylmercury (MeHg) by bacterial activity.1 The anthropogenic perturbation of Hg in the ocean has been recently reported, suggesting an important increase in thermoclines and surface waters in comparison to preanthropogenic conditions.2 Considering the risk to human health of consuming fish products with high MeHg content, special attention has been paid to the analyses of the edible part of the fish (muscle). However, the mechanisms of bioaccumulation, transformation, and metabolization, in general, remain uncertain. Hg speciation approaches, extended to organs different than muscle, revealed that Hg species distribution seems to be organ and species-specific dependent.3−7 It has been reported that the uptake and trophic transfer is more efficient for MeHg in comparison with the inorganic form.6,8−10 In general, the organomercurial species is stocked in the muscle, which exhibits a low turnover.7,9 In contrast, liver, brain, and kidney have been related with potential detoxification mechanisms due to the © XXXX American Chemical Society

Received: July 24, 2015 Revised: September 16, 2015 Accepted: September 23, 2015

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DOI: 10.1021/acs.est.5b03587 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology

and MeHg diet, respectively (Gentès et al., 2015). The fish daily intake provides more valuable information than the net concentration in the supplied food. However, the former is not currently used in previous studies of the isotopic signature in fish, which makes the comparison of the results difficult. More details about the preparation and the provided food pellets are included in the Supporting Information. Fish were harvested after 0, 7, 25, and 62 days of exposure, and skeletal muscle, brain, and liver (see Supporting Information for more details) were collected and stored at −20 °C before analyses. Hg concentration in water of the different aquariums after 7, 25, and 62 days of exposure is summarized in Table S1. The comparison between the Hg level in water and food evidence that diet is the main Hg source for the animals. It should be mentioned that no fish mortality and no health impact on external inspection was observed during the whole experiment (Gentès et al., 2015). Hg Speciation and Stable Isotope Analyses. All the samples (muscle, brain, liver, feces, and food) were digested with TMAH (tetramethylammonium hydroxide) in an analytical microwave (Discover SP-D, CEM) for speciation purposes. Quantification of Hg species was carried out by isotope dilution analysis, using a GC-ICP-MS (Thermo Electron GC, model Trace Ultra, coupled to a Thermo Electron ICP-MS X series XII), as detailed elsewhere.40,41 Several reference materials of biological origin were used for method validation (Table S2). Approximately 0.2 g of sample (freeze-dried) was digested with 3−5 mL of HNO3 acid (depending on the Hg content), after a predigestion step (with 5 mL HNO3 overnight at room temperature), by using HPA (High Pressure Asher),42 for Hg stable isotope analysis. The obtained extracts were stored at −20 °C before analysis. Hg isotopic composition was determined in all samples by using CVG-MC-ICP-MS, as detailed elsewhere.23 Analytical uncertainty was determined as the largest 2SD corresponding to the sample or to the measurement of the UM-Almadén secondary standard during each session (δ202Hg = −0.52 ± 0.16, Δ 199 Hg = −0.03 ± 0.06, n = 31). Previously characterized23,43 BCR-CRM 464 fish muscle was also used as secondary standard (Table S3).

(bio)transformation,24−26 interorgan distribution,28,29 and excretion,31−33 among others. On the other hand, MIF provides complementary information on physical and chemical processes involved in Hg cycling. Even if the processes that induce MIF are not fully elucidated, it has been principally associated with photoreactions and most of the performed studies until present coincide with the absence of MIF signature modification during biological processes.24−26,29,31−33 Therefore, the Hg isotopic system can be considered as a double tracer for both sources and processes. Hg isotopic fractionation response in different fish species have been recently reported by Kwon et al.28,29 They address the role of Hg concentration and the feeding rate in the resulting Hg isotopic signature. The establishment of a clear link between Hg metabolic pathways and the (mass dependent) isotopic fractionation associated with them represents a big challenge. It is principally due to the unawareness about details concerning the mechanisms that constitutes Hg complex biological processes. Even if, in the last years, the structural identity of some biomolecules containing Hg has been elucidated,34−38 providing valuable information about its potential role in Hg detoxification, there is still a lack of knowledge of Hg pathways in living organisms. The main aim of this work is the investigation of the specific iHg and MeHg pathways in a model fish, Danio rerio by a novel approach based on Hg speciation and isotopic fractionation combined with complementary information (metallothioneins (MTs) concentration, electronic microscopy observation, and gene expression, Gentès et al. 2015, companion publication). The animals were fed during 12 months with food pellets prepared from marine seafood bycatch products containing Hg at environmentally relevant concentrations. Two groups of animals raised in parallel were switched after 12 months to a different diet, enriched in MeHg and iHg, for 62 days.

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EXPERIMENTAL SECTION Experimental Design. A detailed description of the experimental design is included in the Supporting Information. Briefly, adult Danio rerio (body weight, 0.6 ± 0.1 g ww; standard length, 30 ± 2 mm; n = 290; low-MeHg diet condition) were simultaneously grown under different dietary conditions during 62 days, after being in the laboratory for 3 weeks to avoid stressful situations produced by environmental change. Fish were separated into three groups, each exposed to a different diet: (1) commercial fish pellets prepared from marine bycatch products (further named low-MeHg diet); (2) diet enriched in MeHg (further named MeHg diet), and (3) diet enriched in iHg (further named iHg diet). While the Hg concentration in the low-MeHg diet was 60 ± 10 ng Hg g−1, mainly constituted by MeHg (80%), in the iHg and MeHg diets, total Hg concentration was 11920 ± 540 and 11580 ± 450 ng g−1, where MeHg represents 1 and 95%, respectively. It should be mentioned that the selection of the Hg (species) exposure level is crucial in such in vitro studies. The choice of dietary conditions was based on the exposure concentrations used in previous studies in which the same fish species were grown under analogous conditions.39 It facilitates the comparison and the interpretation of the obtained data because the deconvolution of the Hg mass dependent fractionation signature and its association to metabolic processes represents a big challenge. The supplied diets correspond to a daily intake of 1, 176, and 171 ng Hg per fish for the groups exposed to low-MeHg, iHg,

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RESULTS AND DISCUSSION Hg Isotopic Characterization of Fish Diet. As previously mentioned, the commercial food pellets are constituted by marine bycatch products and other additives. The Hg isotopic composition in pellets is therefore considered a reflection of such seafood constituents. The obtained positive MIF (Δ199Hg) value (1.13 ± 0.12‰) is comprised in the reported MIF signature range of marine fish,22,23,32,33 as well as the Δ199Hg/Δ201Hg ratio (1.22 ± 0.04‰). It is in total agreement with Hg isotopic composition in other commercial pellets.28 The Hg-enrichment of commercial pellets by spiking iHg and MeHg produces a shift on the Hg isotopic composition of the pellets (Table S4), matching the isotopic signature (MDF and MIF) of iHg (NIST 3133) and MeHg (STREM) standards.43 Response to Low-MeHg Diet Exposure. As mentioned above, the low-MeHg group was fed with pellets prepared from marine bycatch products, principally constituted by MeHg (80%). It should be noticed that once in the laboratory and before the beginning of the experiment, the fish were fed with the same food as the fish breeder in order to minimize fish stress. This means that animals grown under low-MeHg dietary B


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Figure 1. Hg species distribution in muscle, liver, brain and feces under different dietary conditions after 0, 7, 25, and 62 days of exposure; (a) low MeHg, (b) enriched in MeHg and (c) enriched in iHg. The values corresponding to Hg concentration (ng g−1) in each organ under the three different dietary conditions are shown in the top of each bar.

perfectly match the value of the supplied food (δ202 Hg, 0.08 ± 0.16 ‰; Δ199Hg, 1.13 ± 0.12 ‰), regardless of their noticeable differences on Hg species distribution/concentration. The Hg species specific isotopic signature in animals exposed to low-MeHg pellets was not experimentally determined due to the low Hg species concentration. However, it can be estimated from the obtained data. Although the processes that leads to MDF and MIF are not completely understood, most of the experimental studies report an absence of MIF in biological processes (Hg methylation/demethylation, transport, etc.)24−26,29,31−33 independently on the extension of the induced MDF. The isotopic composition in the muscle and the food pellets is a mixture of the MDF and MIF of each Hg species. Fish muscle is considered as an important pool of MeHg, which exhibits a low depuration rate.7,9 If we assume that the iHg source in the muscle is exclusively coming from the diet, the isotopic composition of both species in the unamended pellets can be estimated by solving the isotope mass balance equations:

conditions were exposed to the same pellets 12 months before the experiment started (t = 0 days). Under low-MeHg conditions, Hg species concentration remains stable in all the organs, except at 62 days. The observed increase matches with the augmentation of Hg level in water of low-MeHg aquariums (detailed in the Supporting Information). MeHg was the principal species in all the analyzed organs of fish grown under low-MeHg conditions (Table S5 and Figure 1). The high bioaccumulation factor (BAF; Table S6) under low-MeHg dietary conditions, in comparison with Hg-enriched conditions, as well as the low Hg concentration in feces, illustrates the efficient accumulation of the organomercurial species. This accumulation is higher in muscle (more than 90%), followed by brain and liver. In the liver, a slight decrease of MeHg percentage is observed (from 76 to 63%), as opposed to muscle and brain, where the species distribution remains stable during the experimental period. A homogeneous Hg isotopic composition is observed in the organs of animals exposed to low-MeHg diet (Table S4 and Figure 2a). MDF and MIF signatures in brain, liver, and muscle C


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Figure 2. Hg Isotopic composition (MDF right panel, MIF left panel) on muscle, brain, liver, and feces of animals exposed to (a) low MeHg-diet, (b) MeHg-enriched diet, and (c) iHg-enriched diet.

This kind of estimation has been successfully used on the deconvolution of MIF signature in urine33 and human hair,31,32 which allows determining the contribution of Hg exposure sources. Regarding MDF, it is known that it can be originated/ affected by biological processes,22,24,26,32,33 and as a consequence being therefore less used on such models. However, considering the high accumulation efficiency of MeHg in fish muscle, the low Hg turnover of this organ and that the MDF signature perfectly matches the supplied food in this and previous studies,28 the estimation of the δ202iHg and δ202 MeHg in the unamended pellets (δ202 iHgCfood and δ202 MeHgCfood, respectively) is estimated by solving the following isotope mass

Δ199Hg Cfood = Δ199MeHg CfoodxfMeHgfood + Δ199iHg Cfoodx (1 − fMeHgfood )

(1)

Δ199Hg muscle = Δ199MeHg CfoodxfMeHgmuscle + Δ199iHg Cfoodx (1 − fMeHgmuscle )

(2)

where f MeHg represent the fraction of MeHg in the sample, Δ199 MeHgCfood and Δ199 iHgCfood are the MIF signature in the unamended pellets of MeHg and iHg, respectively. Δ199HgCfood and Δ199Hgmuscle are the MIF signature measured in food and muscle samples, respectively. D


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been also reported in human brain of individuals exposed to organomercurial compounds.49 This hypothesis is in good agreement with the kinetic MIF variation in the feces of the animals exposed to low-MeHg pellets (Figure 2a). The MIF isotopic signature increases from 0.26 ± 0.06 ‰ at 7 days, which match the estimated Δ199iHg in unamended pellets to 0.67 ± 0.06 ‰ at 62 days, probably due to the increase of the contribution of the ingested MeHg (Δ199 MeHgCfood: 1.33 ‰). The interpretation of the MDF data is much more complex because, contrary to MIF that is preserved during metabolism, it can be largely affected by such processes.23,25−27,31 The different metabolic pathways in which Hg is involved remain unknown, as well as the extension of the fractionation (MDF) induced by such biological processes. It has recently been reported that urinary excreted Hg is constituted by lighter isotopes (δ202Hg) in comparison with the body as a result of a MDF due to metabolic processes.33 A similar trend is registered in the group of animals exposed to low-MeHg diet of the current study when comparing the MDF signature in feces and the fish body (Figure 2a). According to the contribution of the different sources of iHg excreted by the animals exposed to low-MeHg diet, it is initially constituted by the ingested iHg from the food pellets. The MDF signature of this species in food is estimated at −0.64 ‰ (δ202Hg); however, the feces exhibits a much lighter MDF value. It seems to be the consequence of different metabolic processes that results in a net diminishment of δ202Hg values. Despite MIF values of feces clearly suggesting that at 7 and 25 days the main source of iHg is the ingestion, there is a noticeable MDF decrease in the same period (7−25 days). It should be considered that animals exposed to low-MeHg were fed with the same diet before the day defined at 0 for this experiment (as detailed in the Experimental Section). Therefore, this variation could reflect the metabolisation/excretion of Hg accumulated for longer periods. It is interesting to notice that the ingested iHg is not expected to be involved in fish methylation.3 The occurrence of Hg methylation in vivo by the action of intestinal bacteria in fish remains controversial, but recent studies in tilapia by using isotopically enriched tracers revealed that Hg methylation extension oscillates between 0.67 and 1.60% of the ingested iHg.3 Thus, even if it takes place (under the experimental conditions of the current study), its influence on the isotopic fractionation should be negligible. MDF signature in feces is therefore the result of different intermediate steps from uptake and transport, before excretion. It has been recently proposed that its transport occurs via a carrier-mediated transcellular mechanism50 with the participation of divalent cation transporters, amino acid transporters, and even some organic anion transporters, as proposed by Zalups and Ahmad.51 The remarkable decrease of δ202Hg from 7 to 25 days, accompanied by a punctual rise of Hg excretion (Figure 2a, Table S4), as well as a specific increase of iHg in liver and brain suggest an alteration on the fish metabolism. It coincides with the activation/beginning of MeHg demethylation according to the estimation of f values. At 62 days, according to the iHg sources estimation in feces, there is an important contribution (approximately 35%) of iHg resulting from MeHg demethylation. The estimated δ202 MeHg (0.25 ‰) is higher than the δ202iHg (−0.64 ‰) in the unamended pellets. Therefore, the observed MDF increase from 25 to 62 days in feces could be related with the excretion of demethylated MeHg even if the

balance equations (eqs 3 and 4). The MDF values were also confirmed by other isotopic mass balance equations, as detailed in the Supporting Information. δ 202 Hg Cfood = δ 202 MeHg CfoodxfMeHgfood + δ 202 iHg Cfoodx (1 − fMeHgfood )

(3)

δ 202 Hg muscle = δ 202 MeHg CfoodxfMeHgmuscle + δ 202 iHg Cfoodx (1 − fMeHgmuscle )

(4)

The estimated isotopic composition in food pellets for iHg and MeHg were Δ199iHgCfood 0.31‰, δ202iHgCfood −0.64‰ and Δ199 MeHgCfood 1.33‰, δ202 MeHgCfood 0.25‰. In general, a quite homogeneous species distribution and isotopic pattern is observed in all the analyzed organs. Noticeable differences regarding the isotopic signature are exclusively registered in the excretion (feces), where both MDF and MIF are lower than the rest of the body, which perfectly matches the supplied food. As detailed in the Experimental Section, unfortunately, speciation analyses were not performed on feces samples of animals exposed to unamended pellets due to mass limitation. Contrary to MeHg, that is the main species excreted by hair,44 in urine and feces it is principally evacuated as iHg,45−47 as confirmed by the investigation in vivo of tilapia (Oreochromis niloticus) dietary exposed to Hg isotopically enriched species.3 As recently reported in human urine, the excreted iHg should represent a mixture of iHg-diet and MeHg-diet demethylation derived.33 Regarding MeHg demethylation by fish, it is debated in some experiments that such species transformation process has not been observed,3 which contrast with significant (hepatic) demethylation observed in Danio rerio39 and marine mammals.15,14 Therefore, both contributions will be consequently reflected on the feces isotopic composition as expressed in eq 5: Δ199Hg feces = Δ199iHg Cfoodxf + Δ199iHgdemethx(1 − f )

(5)

where Δ iHg Cfood is the Δ iHg estimated in the unammended pellets. Assuming that no MIF occurs during biotic demethylation,24,26 Δ199iHgdemeth is equal to Δ199 MeHg estimated in the non Hg enriched pellets. Therefore, if we adopt that MIF is preserved during biological transport27 and excretion processes,31−33,28,29 the fraction of dietary iHg ( f) can be estimated by using eq 6: 199

199

Δ199Hg feces = Δ199iHg Cfoodxf + Δ199MeHg Cfoodx(1 − f ) (6)

The obtained f values are 1, 0.94, and 0.65 for 7, 25, and 62 exposure days, respectively. It suggests that the contribution of iHg in the feces from MeHg demethylation is almost negligible at the beginning of the experiment, increasing up to 35% of the total Hg excreted after 62 days. MeHg demethylation processes in vivo are not completely elucidated. It has been suggested for marine mammals, species where such processes have been largely studied, that demethylation takes place principally in the liver.15,3,48 Among other factors, it is considered dependent on the individual maturity and MeHg concentration levels.3,48 It is therefore probable that the rise of Hg levels leads to the beginning of the hepatic MeHg demethylation processes. It should be mentioned that MeHg demethylation in vivo has E


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to the MeHg demethylation followed by the precipitation as tiemmanite (HgSe).13−15 However, in tilapia, it has been recently demonstrated that a diminishment of the MeHg percentage in liver is not a result of MeHg demethylation, but of transport of this species from liver to other organs.3 The transfer of MeHg from liver to muscle in field studies, by using yellow perch, has also been reported.47 Specifically in the case of zebrafish, previous studies reported a MeHg decrease, exclusively in liver, attributed to demethylation in this vital organ.39 However, despite being carried out with the same fish species under the same experimental conditions, in the current work the percentage of MeHg in liver remains around 90%. The repression of genes involved in cellular protection at 7 days suggests significant early toxic damages to cells of MeHg. It is confirmed by electron microscopy observation that reveals severe damages to hepatic tissues (Gentès et al., 2015). The lack of an efficient MeHg demethylation mechanism seems to be a consequence of damage, already induced during the first 7 days, by the MeHg concentration levels. The dissimilar behavior between the animals under similar conditions may be explained by the fish origin, wild and laboratory field, as described in Gentès et al. (companion paper). The homogeneous isotopic composition observed in all the organs, matching the signature of the supplied food is in good agreement with the results published by Kwon et al.28,22 Juvenile and mature freshwater fish, exposed to different MeHg level containing food show an indistinguishable isotopic composition between organs, irrespectively of the differences on Hg content, matching the isotopic signature of the supplied food.28 A similar trend is also observed in marine fish (amberjack, Seriola dumerili), when fed with a high-MeHg diet. The direct transfer of the food isotopic composition to the different analyzed organs has been attributed to the efficient accumulation of MeHg, the major species in the supplied food.28,29 In contrast, the ingestion of a low-MeHg diet, as well as feeding rate changes, could lead to an incomplete turnover and mixing of MeHg, producing a partial shift of Hg isotopic signature compared to the food source.22 It is interesting to notice that the isotopic composition shift observed in marine and freshwater fish at different Hg (principally as MeHg) level diets exhibits a gradual mixing between the food sources.28,22 For example, the consumption of tuna (Thunnus atlanticus) diet by amberjack leads to a shift to the new food source after 30 days of exposure.22 It is first observed in the vital organs and finally in the muscle.22 The most efficient transfer to organs such as liver and brain is supposed to be through blood, where MeHg is principally bound to hemoglobin, as recently demonstrated.35 However, in our study, the consumption of a MeHg diet produces an isotopic re-equilibration observable after 7 days of exposure. In contrast with previous studies,28,29 where the isotopic signature shift is first observed in vital organs and delayed in muscle, in our experiment after 7 days of MeHgexposure, all the analyzed organs exhibit an indistinguishable isotopic fractionation. Evidently, such differences can be attributed, among others, to the specific metabolism of the respective animal species. In general terms it is difficult to make a comparison with previous studies considering the differences of the experimental conditions (animal species, wild or fish line, age, and exposure concentration and duration).

extension of the MDF caused by this reaction cannot be determined. Demethylation is well recognized to cause an enrichment of the produced iHg in lighter isotopes in comparison to MeHg.26 However, such an increase in δ202 MeHg value is not observed in the analyzed organs likely due to the following aspects. The concentration of iHg originally from demethylation, approximately 30 ng g−1 at 62 days, is much lower than the MeHg in the organs. Therefore, the enrichment in MeHg (MDF), assuming that demethylation takes place exclusively in one organ, is not perceptible. As the δ202Hg is the net result of the contribution of δ202Hg of both species (iHg and MeHg), if one of them is enriched in lighter isotopes in the same extension that the other one is enriched in heavier ones, the isotopic mass balance is preserved, explaining no variations in δ202Hg. In addition, the MDF signature of the excreted iHg produced by demethylation is also influenced by the multiple transport steps before excretion. The lack of knowledge of the metabolic pathway of Hg species hampers a complete deconvolution of the resulting MDF. Response to MeHg-Diet Exposure. Diet enrichment with MeHg leads to an increase of such species with the incubation time in all the analyzed organs. It represents the main species in all cases (higher than 90%), being preferentially accumulated in brain followed by liver. Under this diet condition, Hg concentration in feces is much lower than in the analyzed organs, being excreted as MeHg. Regarding Hg isotopic composition, all organs experiment a decrease of approximately 1‰ of MDF and MIF values after 7 days MeHg-diet exposure. It results in a homogeneous Hg isotopic composition of all organs, that matches with the isotopic composition of the supplied food (δ202Hg, −0.84 ± 0.11 ‰; Δ199Hg, 0.03 ± 0.07‰; Figure 2b). This rapid reequilibration of the Hg isotopic composition of the internal organs to the new MeHg-food source seems to be a consequence of the high bioaccumulation rate of this organomercurial species. It should be noted that the isotopic signature of MeHg-food is preserved after 7 days, irrespectively of the noticeable increase of Hg concentration and BAF differences in each organ. The kinetic increase of the MeHg BAFs in the analyzed organs (Table S6) suggests that dietequilibrium had not been raised yet. Hg concentration in the different organs of animals exposed to MeHg-diet is in good agreement with values previously reported by Gonzalez et al. under analogous conditions.39 However, there are noticeable differences related to Hg species distribution in liver. In the current study, MeHg represents more than 90% of the total Hg in liver contrasting to the previous report of a decrease of MeHg from 66 to 36% during the same exposure period.39 The high percentage of this organomercurial species in liver (Figure 1), which also remains stable during the exposure time, does not indicate a significant hepatic MeHg demethylation. The percentage of iHg in brain and muscle tissue is lower than 5%. Regarding in vivo demethylation process, there is no agreement about the organ(s) in which it takes place. Even if in some living organisms the increase of iHg in brain of animals exposed to MeHg has been attributed to potential MeHg demethylation in brain,11,12 the hypothesis of demethylation in other organs followed by transport and accumulation in the brain is not discarded.11 In general terms, liver is considered as a key organ for Hg detoxification, associated with the binding of iHg to MTs34 and F


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Hg contained in liver is almost exclusively originally from the supplied iHg-enriched diet, the variations on MDF are not due to the contribution of an additional iHg source, but to metabolic processes. The progressive enrichment of liver in Hg heavier isotopes (MDF, δ202Hg from −0.79 to −0.22 ‰) after 7 days could be associated with iHg redistribution to other organs. This hypothesis is confirmed by the estimated δ202iHg values in the analyzed organs (Table S7). The enrichment of liver in heavier δ202iHg coincides with the enrichment of brain in lighter ones, meanwhile the isotopic composition of this species remain stable in the muscle. It is well established that transport processes induce MDF, causing enrichment in heavier isotopes on the source.27 Therefore, this result can be understood as a release of iHg from liver to the brain, after the initial accumulation in the hepatic organ. It is in good agreement with the MIF data, which reveals that liver is the target organ for iHg, followed by brain, under such experimental conditions. These results are in agreement with the biomarkers response observed in the analyzed organs (Gentès et al., 2015). The fact that iHg is preferentially accumulated in liver could be associated with the detoxification capacity of such organ. A higher basal level of MT in liver than in brain and muscle is observed, considering the high affinity of such proteins for iHg,34,53 higher iHg concentrations are needed to induce MT synthesis in liver. A higher base level of some genes (as gpx4a involved in oxidative stress response), and few genetic responses (sod gene) showed the efficiency of liver as a detoxification organ. In brain, the induction of mt2 gene expression after 7 days of exposure reveals the ability of such organs to defend against iHg through the MT. It is interesting to notice that previous studies revealed a delay on the accumulation of dietary MeHg in muscle.7,29,47 It is attributed to a rapid equilibration in vital organs like liver, brain and kidney, contrasting with a slower turnover in muscle. In our study, such pattern is observed in animals exposed to iHg, demonstrating that it is not exclusive of the organomercurial species. According to isotopic composition and Hg distribution, muscle seems to be the organ less impacted by iHg, receiving the lowest amount of such species, principally accumulated in liver, followed by brain. However, the genetic response is more noticeable than in the other organs, principally observed after 62 days of exposure. It is related to its ability to react against iHg through the induction of genes involved in defense mechanisms (oxidative stress, mitochondrial metabolism, nervous transmission to protect cells). It should be noticed that electron microscopy analyses of muscle did not reveal important tissue damages (Gentès et al., 2015). The MDF pattern achieves a homogeneous signature after 62 days of exposure. It is undoubtedly due to the reached equilibrium as a consequence of the ability of Hg species to bind biomolecules facilitating its transport between the different organs/systems. The resulting value (δ202Hg ∼ −0.20 ‰) is slightly higher than the supplied food, probably due to an enrichment in heavier isotopes in the fish body in comparison to the excretion (in the following section). Excretion under Hg-Enriched Diets. Hg excretion processes has been traced by stable isotopes in recent studies, all of them related to human Hg exposure.30−33,44 The excretion has been tracked in urine and individual’s hair, where Hg is present in the form of iHg and MeHg, respectively,.44,54,46,47 The Hg isotopic pattern in such samples allows the discrimination of the different Hg sources, such as

The rapid isotopic re-equilibration observed in the current experiment suggests a very efficient MeHg accumulation. It is in perfect agreement with the response of several biomarkers (Gentès et al., 2015). Response to iHg-Diet Exposure. Concerning iHg dietary enrichment, the concentration of this species increases in all the organs with the exposure time (Table S5 and Figure 1). In contrast to MeHg conditions, the distribution of both Hg species gradually changes over the time. The percentage of MeHg is progressively reduced up to 50 and 10% in muscles and brain, respectively, after 62 days. In liver, the organ which accumulates the highest amount of Hg under iHg dietary conditions (3568 ± 262 ng·g−1 at 62 days), the percentage of iHg increases sharply after 7 days of exposure, remaining constant until the end of the experiment. Hg is exclusively excreted as iHg exhibiting a much higher concentration than in the analyzed organs. These bioaccumulation patterns are in good agreement with previous observations in freshwater tilapia.3,52 Under iHg-dietary conditions, a dissimilar Hg isotopic composition was observed in the different organs, contrasting to MeHg conditions. The kinetic MIF variation differs between the analyzed organs (Figure 2c). Assuming that there is no Hg methylation/demethylation under the experimental conditions, the Δ199Hg signature of each organ can be deconvoluted as (eq 7). This hypothesis is validated by the total coherence between the measured and the modeled data (Supporting Information, Figure S2) Δ199Hgorgan = Δ199MeHg iHg − foodx f + Δ199iHg iHg − foodx(1 − f )

(7)

where Δ199iHgiHg‑food and Δ199 MeHgiHg−food are the MIF isotopic signature of iHg and MeHg estimated in the iHgenriched food respectively (Supporting Information), f is the fraction of MeHg in the sample determined by speciation analyses. A particular trend is observed in the liver of iHg-exposed animals (Figure 2c). Corresponding to the highest iHg concentration, at 7 days exclusively this organ reflects the MIF isotopic signature of the supplied food, which remains stable until the end of the experiment. The specific quick shift of the initial liver MIF into the iHg-diet one, indicates that it is the target organ of the ingested iHg. It is supported by the noticeable shift (0.75 ‰ δ202Hg values) of the MDF signature specifically experimented by liver during the first 7 days of iHg exposure. The resulting MDF signature is slightly lower than in the supplied iHg-diet, confirming that ingested iHg is preferentially accumulated in the hepatic organ. In contrast to liver, where the MIF signature remain unalterable from 7 days until the end of the experiment, the trend in brain and muscle (Figure 2c) shows a decrease from the initial value toward the MIF signature of the supplemented iHg-diet. Such variation is more accentuated in brain than in muscle, and suggests that after liver, dietary iHg is favorably accumulated in the former. It is in perfect agreement with the hypothesis established for the origin of iHg in the group exposed to low-MeHg diet, confirming that diet is the principal source of iHg. Regarding MDF, there are noticeable differences on the kinetic behavior of the analyzed organs. Considering that MIF is not affected by metabolic processes, which evidence that the G


Article

Environmental Science & Technology MeHg-rich diet,30,32,33 dental amalgams33 and occupational exposure.31−33 It has been recently reported that urinary excreted Hg is constituted by lighter isotopes in comparison with the body.33 The opposite isotopic MDF signature in hair, enriched in heavier isotopes, and urine in lighter ones, is attributed to MDF induced by metabolic processes.33 In the current study, the trend of excretion of lighter isotopes (δ202Hg) is observed exclusively in feces of animals of the group exposed to low-MeHg diet (discussed above). In contrast, the isotopic signature, MDF and MIF, in feces of animals exposed to iHg and MeHg, matched the supplied food. Under iHg-dietary conditions the proportion of Hg excreted by fish is much higher than under MeHg and low-MeHg conditions, in total agreement with previous studies that concludes a faster excretion of iHg than the organomercurial species by fish.55 It is coherent with the obtained BAFs, endorsing the low bioaccumulation of iHg due to its excretion. As previously mentioned, the kinetic isotopic variation in the liver of such organisms could be related with the mobilization/ excretion of lighter Hg isotopes. However, due to the elevated rate of excretion, the potentially iHg fractionated portion in feces is not perceptible, and it matches the isotopic pattern of the supplied food. Despite the similar Hg speciation in low-MeHg and MeHgenriched food pellets (dominated by MeHg in more than 80%), divergences on the isotopic signature of the supplied diet and the excretion is observed only in samples of animals exposed to low-MeHg diet. MDF variations are not observed in the excretion of animals exposed to MeHg-diet, probably due to the overlapping of the lighter metabolized Hg with the high proportion of nonmetabolized dietary MeHg. It should be noticed that isotopic fractionation is principally observed in the excreted Hg under low-MeHg dietary conditions. In contrast to previous studies of Hg excretion, where principally MIF signature is exploited, in the current work the kinetic variation of MDF provide precious information. In total coherence with the “classical” Hg sources identification in feces by MIF, the variations in MDF at 62 days evidence the contribution of iHg from MeHg-demethylation processes. In addition, MDF signature brought information about transport and processes before excretion. Despite the advances on Hg speciation in biological samples, there is still an important lack of knowledge about the different and complex metabolic pathways in which Hg is involved, not only in this fish species, but in living organisms in general. It makes the association of the extension and sense of the isotopic fractionation to the different metabolic steps difficult. However, the convergence of advances in speciation of Hg at a (bio)molecular level and Hg stable isotope fractionation are expected to become a powerful tool in metabolic studies. In summary, the main outcome of this study is the influence of species specific dose−response on the Hg isotopic variation in fish organs. Noticeable differences on the isotopic and speciation patterns between fish exposed to iHg- and MeHgenriched food are observed as a reflex of the dissimilar metabolization of both Hg species. These results are in excellent agreement with the MT extent and genetic response (Gentès et al., companion paper). This work provides valuable information about Hg metabolic pathways and constitutes clear evidence of the vast potential of Hg stable isotopic signature on metabolic studies. It also reveals the challenges associated with the understanding of such

biochemical processes through the isotopic variation and claim for new studies at different (metabolic) levels in order to facilitate its deconvolution/interpretation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b03587. Additional materials as described in the text. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +33 (0)5 40 17 5027 E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the French National Research Agency through ANR-11-CESA-0013 RIMNES Project (Mercury Isotopes Fractionation and NOTCH/apoptosis biomarkers: a new link between Environment and Health). C.F. acknowledges the CSC (Chinese Scholarship Council, N 201204910188) for her PhD grant.

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REFERENCES

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Specific Pathways of Dietary Methylmercury and Inorganic Mercury

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