Accepted Manuscript Title: Dietary selenomethionine influences the accumulation and depuration of dietary methylmercury in zebrafish (Danio rerio) Author: Heidi Amlund Anne-Katrine Lundebye David Boyle St˚ale Ellingsen PII: DOI: Reference:
S0166-445X(14)00346-4 http://dx.doi.org/doi:10.1016/j.aquatox.2014.11.010 AQTOX 3975
To appear in:
Aquatic Toxicology
Received date: Revised date: Accepted date:
5-6-2014 7-11-2014 13-11-2014
Please cite this article as: Amlund, H., Lundebye, A.-K., Boyle, D., Ellingsen, S.,Dietary selenomethionine influences the accumulation and depuration of dietary methylmercury in zebrafish (Danio rerio), Aquatic Toxicology (2014), http://dx.doi.org/10.1016/j.aquatox.2014.11.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Dietary selenomethionine influences the accumulation and depuration of
ip t
dietary methylmercury in zebrafish (Danio rerio)
an
us
cr
Heidi Amlund1, *, Anne-Katrine Lundebye1, David Boyle2 and Ståle Ellingsen1
1 National Institute of Nutrition and Seafood Research (NIFES), P. O. Box 2029 Nordnes,
M
5817 Bergen, Norway
Ac ce p
te
2E9
d
2 Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada, T6G
*Corresponding author: [email protected], +47 55 90 51 00
1 Page 1 of 34
Abstract Methylmercury (MeHg) is a toxicant of concern for aquatic food chains. In the present study
cr
MeHg toxicokinetics was characterised in zebrafish (Danio rerio).
ip t
the assimilation and depuration of dietary MeHg and the influence of dietary selenium on
us
In a triplicate tank experimental design (n = 3 tanks per treatment group) adult zebrafish were exposed to dietary MeHg (as methylmercury-cysteine) at 5 and 10 µg/g and with or without
an
selenium (as selenomethionine) supplemented to the diets at a concentration of 5 µg/g for eight weeks followed by a four week depuration period.
M
Methylmercury accumulated in muscle, liver and brain of zebrafish; with higher mercury concentrations in liver and brain than in muscle following eight weeks of exposure. In muscle,
d
the mercury concentrations were 3.4 ± 0.2 and 6.4 ± 0.1 µg/g ww (n = 3) in zebrafish fed the
te
5 and 10 µg Hg/g diets, respectively. During the depuration period, mercury concentrations
Ac ce p
were significantly reduced in muscle in both the 5 and 10 µg Hg/g diet groups with a greater reduction in the high dose group. After depuration the mercury concentrations were 2.4 ± 0.1 and 4.0 ± 0.3 µg/g ww (n = 3) for zebrafish fed the 5 and 10 µg Hg/g diets, respectively. Data also indicated that supplemented dietary selenium reduced accumulation of MeHg and enhanced the elimination of MeHg. Lower levels of mercury were found in muscle of zebrafish fed MeHg and SeMet compared with fish fed only MeHg after eight weeks exposure; the mercury concentration in muscle were 5.8 ± 0.2 and 6.4 ± 0.1 µg/g ww (n = 3) for zebrafish fed the 10 µg Hg/g + 5 µg Se/g diet and the 10 µg Hg/g diet, respectively. Furthermore, the elimination of MeHg from muscle during the four week depuration period was significantly greater in the fish fed the diet containing SeMet compared to a control diet;
2 Page 2 of 34
the mercury concentrations were 3.3 ± 0.1 and 4.0 ± 0.3 µg/g ww (n = 3) for zebrafish fed the 5 µg Se/g and the control diets, respectively. In summary, dietary SeMet reduces the
Dietary
exposure,
methylmercury,
selenium,
toxicokinetics,
zebrafish
Ac ce p
te
d
M
an
us
cr
Keywords
ip t
accumulation and enhances the elimination of dietary MeHg in muscle of zebrafish.
3 Page 3 of 34
1. Introduction Mercury, in the form of methylmercury (MeHg), is a toxicant of concern for aquatic food
ip t
chains. In fish, exposure to MeHg has been shown to cause oxidative stress in brain of
cr
Atlantic cod (Gadus morhua) following intraperitoneal injection with MeHg (Berg et al., 2010) and in brain (Berntssen et al., 2003) and liver (Olsvik et al., 2011) of Atlantic salmon
us
(Salmo salar) after dietary exposure. Prolonged dietary exposure to MeHg has also been shown to affect growth, reproduction and neuronal function (Depew et al., 2012). Dietary
an
MeHg is highly bioavailable to fish and known to accumulate in all tissues (Berntssen et al., 2004; Clarkson, 1997), including the brain after traversing the blood-brain-barrier and also
M
muscle (Amlund et al., 2007; Berntssen et al., 2004). In contrast, elimination of MeHg from tissues is slow (Amlund et al., 2007) and as a consequence MeHg biomagnifies through
d
aquatic food chains (Fitzgerald and Mason, 1997; Morel et al., 1998). In aquaculture, the
te
inclusion of marine protein (fishmeal) in fish feed is the dominant source of mercury (and
Ac ce p
predominantly in the form of MeHg) for farmed fish. Hence, there is a considerable interest in identifying methods to be used in industry to decrease the bioaccumulation of MeHg in farmed fish to both reduce the risk of toxicity in fish and to minimise exposure to MeHg in humans.
Selenium is an essential cofactor for a number of selenoproteins, including glutathione peroxidases (GPxs) and thioredoxin reductase (TrxR), which have antioxidant functions (Arteel and Sies, 2001). Selenium is also a possible MeHg antagonist and has been shown to affect the kinetics of MeHg accumulation and tissue partitioning behaviour in fish (Bjerregaard et al., 1999; Bjerregaard et al., 2011; Branco et al., 2012; Dang and Wang, 2011; 4 Page 4 of 34
Deng et al., 2008; Huang et al., 2013; Ringdal and Julshamn, 1985; Sheline and SchmidtNielsen, 1977). However, these effects of selenium on MeHg uptake and elimination are complex and are dependent upon the chemical speciation of selenium. For example, dietary
ip t
selenite (SeO32-), selenocysteine (SeCys; HSeCH2CH(NH2)CO2H) and selenomethionine (SeMet; CH3SeCH2CH2CH(NH2)CO2H) increased the elimination of MeHg from goldfish
cr
(Carassius auratus) in a dose-dependent manner, whereas dietary selenate (SeO42-) had no effect (Bjerregaard et al., 2011). Dietary selenite has also been shown to increase the
us
elimination of MeHg from tissues, including muscle, liver and kidney, of rainbow trout (Oncorhynchus mykiss) (Bjerregaard et al., 1999) and dietary SeMet decreased the
an
accumulation of dietary MeHg in Sacramento splittail larvae (Pogonichthys macrolepidotus)
M
(Deng et al., 2008) and zebrafish (Danio rerio) (Penglase et al., 2014). Interestingly, coexposure to MeHg and SeMet in the diet increased the accumulation of selenium in splittail
d
larvae compared to larvae fed only SeMet, possibly indicating an increased requirement for
te
selenium in MeHg exposed fish (Deng et al., 2008). In contrast to these studies, dietary selenium (selenite, selenite, SeCys or SeMet) did not affect the kinetics of dietary MeHg in
Ac ce p
jarbua terapon (Terapon jurbua); the assimilation efficiency of MeHg was not reduced when the fish were either pre- or co-exposed to selenium (Dang and Wang, 2011). Collectively these studies indicate that dietary selenium may affect both accumulation and elimination of MeHg in fish, and vice versa, however the importance of selenium speciation, the interaction between selenium and MeHg, and the reasons for differences observed between species of fish are still poorly understood.
How selenium affects uptake, elimination and also toxicity of MeHg in fish is unclear. A possible mechanism is the formation of MeHg-selenol compounds; Selenium has been hypothesised to ameliorate MeHg toxicity through ligand exchange with MeHg-complexed 5 Page 5 of 34
thiol residues of proteins (Kahn and Wang, 2009). Conversely, the toxic effects of MeHg have been suggested to be due to a deficiency of selenium combined with an inhibition of selenoproteins as a result of MeHg-selenol complexation (Ralston and Raymond, 2010). For
ip t
example, the activities of GPx and TrxR were reduced in brain and liver of zebra-seabream (Diplodus cervinus) following waterborne exposure to MeHg (Branco et al., 2011; Branco et
cr
al., 2012). Nevertheless, in the digestive tract, the binding of MeHg to selenol groups could also lead to a decreased bioavailability of MeHg and reduced toxicity (Kahn and Wang,
an
us
2009).
The aim of the present study was to characterise the assimilation and depuration of dietary
M
MeHg, and to investigate the influence of dietary SeMet on MeHg bioaccumulation, in zebrafish. Zebrafish is an emerging model in toxicology and fish nutrition research, and have
te
Ac ce p
al., 2008; Liu et al., 2002).
d
been effectively applied to kinetic studies of dietary metals (Bjerregaard et al., 2011; Boyle et
2. Materials and methods
2.1 Experimental animals and study design The feeding trial was performed at the National Institute of Nutrition and Seafood Research, Bergen, Norway. The experiment was approved by the Norwegian Animal Research Authority and performed according to national and international ethical standards. During the pre-exposure acclimation period and during the experiments, fish were housed in a MultiRack System for zebrafish (Aquatic Habitats Inc., Apopka, FL, USA) with continuously aerated and triple-filtered (mechanical, chemical (activated carbon) and biological) 6 Page 6 of 34
recirculating water system (28.5°C, pH 7.5, 500 µS/cm 14h:10h light:dark photoperiod, and 10% daily water exchange). Zebrafish (males and females) were randomly distributed among 15 tanks (9L), in a triplicate
ip t
tank per treatment design and with 33-53 individuals in each tank, depending on the dietary treatment. The zebrafish were fed three times a day, at a total ration of 1.0% of their body
cr
weight daily. Zebrafish were fed the MeHg and/or selenium enriched diets, for eight weeks. A
us
control diet (no added mercury or selenium) was also included in the experimental design. After eight weeks of exposure two dietary groups (5 µg Hg/g diet; and 10 µg Hg/g and 5 µg
an
Se/g diet, respectively) were terminated. Groups fed a 5 µg Hg/g diet were transferred to the control diet, while groups fed the control and 10 µg Hg/g diets were split into six groups each.
M
Half of the groups were fed a control diet, while the other half were fed a 5 µg Se/g diet for four weeks. All groups of fish were kept in 3L tanks (11-15 fish per tank) during the four
Ac ce p
2.2 Experimental diets
te
d
weeks of depuration. The experimental design is summarised in Figure 1.
The experimental diets were produced by adding aqueous solutions of MeHg and/or SeMet to a commercial pelleted zebrafish diet (Aqua Schwarz, Göttingen, Germany). Methylmercury was added to diets as methylmercury-cysteine (MeHg-cys), which was prepared as described earlier (Glover et al., 2009). In brief, a stock solution of methylmercury(II) chloride (Alfa Aesar, Karlsruhe, Germany) was mixed with a stock solution of cysteine (dissolved in water; L-cysteine, Sigma-Aldrich, Seelze, Germany) in a 1:1.2 molar mixture. Selenium was added as SeMet (dissolved in water; Seleno-L-methionine, Sigma-Aldrich). The diets were produced in batches of 10 g with three ml of the appropriate solution (MeHg and/or SeMet) added gradually (1 ml at a time) to 10 g diet while stirring to avoid clumping of the pelleted diet.
7 Page 7 of 34
Following the addition of the solutions, the diets were dried at room temperature in a fume hood until a constant weight of 10 g was obtained (addition of solutions led to no net weight gain). The trays with batches of diet were occasionally shaken to avoid clumping of the
ip t
pelleted diet. The diets were stored at 4°C in the dark until required. Methylmercury alone was added to diets at nominal concentrations of 0, 5 and 10 µg Hg/g. Selenomethionine was
cr
added to one diet at a nominal concentration of 5 µg Se/g, and MeHg and SeMet were both added to one additional diet at nominal concentrations of 10 µg Hg/g and 5 µg Se/g,
us
respectively. The measured total mercury concentration of the diets were 0.08 ± 0.01 µg/g (n = 3) for the control diet, 5.2 ± 0.4 µg/g (n = 3) for the 5 µg Hg/g diet, 9.8 ± 0.5 µg/g (n = 3)
an
for the 10 µg Hg/g, 0.16 ± 0.04 µg/g for the 5 µg Se/g diet and 9.3 ± 0.6 µg/g (n = 3) for the
M
10 µg Hg/g + 5 µg Se/g diet. The measured selenium concentrations were 2.3 ± 0.1 µg/g (n = 3) for the control diet, 2.2 ± 0.5 µg/g (n = 3) for the 5 µg Hg/g diet, 2.3 ± 0.1 µg/g (n = 3) for
d
the 10 µg Hg/g, 4.3 ± 0.1 µg/g for the 5 µg Se/g diet and 4.3 ± 0.2 µg/g (n = 3) for the 10 µg
te
Hg/g + 5 µg Se/g diet. All diets were balanced with cysteine and/or methionine (L-
Ac ce p
methionine, Sigma-Aldrich) to equal the nominal levels in the 10 µg Hg/g + 5 µg Se/g diet.
2.3 Tissue samplings
To guarantee sufficient tissue for measurements of mercury and selenium, tissues (muscle, liver and brain) of n = 3 zebrafish from each tank were pooled. Zebrafish (n = 3 per tank) were sampled at weeks 2 and 8 during the exposure period and at the end depuration period (Week 12). In addition zebrafish were sampled at the start of the feeding trial (n = 3, replicate samples). The zebrafish were sampled randomly and immediately killed by an overdose of MS-222 (0.5 g/L; ethyl 3-aminobenzoate methanesulfonate; Sigma-Aldrich). Length and weight of the zebrafish were recorded and brain, liver and muscle were excised. Pooled samples (n = 3 fish per pool) were kept on ice during sampling, and then stored at -20 °C until 8 Page 8 of 34
further analyses. For some zebrafish and time points only muscle was analysed as muscle is an important site of accumulation of mercury (Berntssen et al., 2004) and of importance
ip t
within aquaculture.
cr
2.4 Mercury and selenium determination by inductively coupled plasma mass spectrometry
Total mercury and selenium were determined in diets and samples by inductively coupled
us
plasma mass spectrometry (ICPMS) after microwave assisted decomposition as previously described (Julshamn et al., 2007). In brief, the samples were digested in 65% nitric acid (2 ml;
an
Suprapur, Merck, Darmstadt, Germany) and 30% hydrogen peroxide (0.5 ml; Merck) using a microwave digestion system (Ethos 1600; Milestone, Sorisole, Italy). The solutions were
M
diluted to 10 ml with deionised water (>17 MΩ/cm; Nanopure System; Barnstead, Dubuque, IA, USA). Total mercury and selenium concentrations in all samples were determined by
d
ICPMS (Agilent ICPMS 7500c; Yokogawa analytical systems, Tokyo, Japan) equipped with
te
an autosampler (ASX-500; CETAC Technologies, Omaha, NE, USA). Data were collected
Ac ce p
and processed using the Agilent Chemstation ICPMS software (Agilent Technologies, Palo Alto, CA, USA). Rhodium was used as an internal standard to correct for any drift of the instrument. The accuracy of the analytical method was assessed by analysis of two certified reference materials; lobster hepatopancreas (TORT-2; National Research Council Canada, Ottawa, Ontario, Canada; for mercury; certified value 0.27 ± 0.06 µg Hg/g; obtained value 0.27 ± 0.00 µg Hg/g, n = 2; for selenium; certified value 5.63 ± 0.67 µg Se/g; obtained value 4.91 ± 0.18 µg Se/g, n = 2) and oyster tissue (SRM 1566b; National Institute of Standards and Technology, Gaithersburg, MD, USA; for mercury; certified value 0.0371 ± 0.0013 µg Hg/g; obtained value 0.032 ± 0.001 µg Hg/g, n = 4; for selenium; certified value 2.06 ± 0.15 µg Se/g; obtained value 2.05 ± 0.06 µg Se/g, n = 4). The limit of quantification (LOQ) of the method is 0.005 and 0.01 µg/g for mercury and selenium, respectively. 9 Page 9 of 34
2.5 Statistical analyses The data presented were evaluated statistically using Statistica® (Ver. 10; StatSoft, Tulsa,
ip t
OK, USA). All data sets were first tested for normality (Shapiro-Wilk test) and homogeneity
cr
of variances (Levene’s test). To detect significant differences between treatment groups, data sets of fish weight, and mercury and selenium concentrations in muscle, liver and brain were
us
analysed using one-way analysis of variance (ANOVA) with post-hoc Tukey’s honestly significance test. For all analyses a 95% confidence interval was applied with a level of p
S0166-445X(14)00346-4 http://dx.doi.org/doi:10.1016/j.aquatox.2014.11.010 AQTOX 3975
To appear in:
Aquatic Toxicology
Received date: Revised date: Accepted date:
5-6-2014 7-11-2014 13-11-2014
Please cite this article as: Amlund, H., Lundebye, A.-K., Boyle, D., Ellingsen, S.,Dietary selenomethionine influences the accumulation and depuration of dietary methylmercury in zebrafish (Danio rerio), Aquatic Toxicology (2014), http://dx.doi.org/10.1016/j.aquatox.2014.11.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Dietary selenomethionine influences the accumulation and depuration of
ip t
dietary methylmercury in zebrafish (Danio rerio)
an
us
cr
Heidi Amlund1, *, Anne-Katrine Lundebye1, David Boyle2 and Ståle Ellingsen1
1 National Institute of Nutrition and Seafood Research (NIFES), P. O. Box 2029 Nordnes,
M
5817 Bergen, Norway
Ac ce p
te
2E9
d
2 Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada, T6G
*Corresponding author: [email protected], +47 55 90 51 00
1 Page 1 of 34
Abstract Methylmercury (MeHg) is a toxicant of concern for aquatic food chains. In the present study
cr
MeHg toxicokinetics was characterised in zebrafish (Danio rerio).
ip t
the assimilation and depuration of dietary MeHg and the influence of dietary selenium on
us
In a triplicate tank experimental design (n = 3 tanks per treatment group) adult zebrafish were exposed to dietary MeHg (as methylmercury-cysteine) at 5 and 10 µg/g and with or without
an
selenium (as selenomethionine) supplemented to the diets at a concentration of 5 µg/g for eight weeks followed by a four week depuration period.
M
Methylmercury accumulated in muscle, liver and brain of zebrafish; with higher mercury concentrations in liver and brain than in muscle following eight weeks of exposure. In muscle,
d
the mercury concentrations were 3.4 ± 0.2 and 6.4 ± 0.1 µg/g ww (n = 3) in zebrafish fed the
te
5 and 10 µg Hg/g diets, respectively. During the depuration period, mercury concentrations
Ac ce p
were significantly reduced in muscle in both the 5 and 10 µg Hg/g diet groups with a greater reduction in the high dose group. After depuration the mercury concentrations were 2.4 ± 0.1 and 4.0 ± 0.3 µg/g ww (n = 3) for zebrafish fed the 5 and 10 µg Hg/g diets, respectively. Data also indicated that supplemented dietary selenium reduced accumulation of MeHg and enhanced the elimination of MeHg. Lower levels of mercury were found in muscle of zebrafish fed MeHg and SeMet compared with fish fed only MeHg after eight weeks exposure; the mercury concentration in muscle were 5.8 ± 0.2 and 6.4 ± 0.1 µg/g ww (n = 3) for zebrafish fed the 10 µg Hg/g + 5 µg Se/g diet and the 10 µg Hg/g diet, respectively. Furthermore, the elimination of MeHg from muscle during the four week depuration period was significantly greater in the fish fed the diet containing SeMet compared to a control diet;
2 Page 2 of 34
the mercury concentrations were 3.3 ± 0.1 and 4.0 ± 0.3 µg/g ww (n = 3) for zebrafish fed the 5 µg Se/g and the control diets, respectively. In summary, dietary SeMet reduces the
Dietary
exposure,
methylmercury,
selenium,
toxicokinetics,
zebrafish
Ac ce p
te
d
M
an
us
cr
Keywords
ip t
accumulation and enhances the elimination of dietary MeHg in muscle of zebrafish.
3 Page 3 of 34
1. Introduction Mercury, in the form of methylmercury (MeHg), is a toxicant of concern for aquatic food
ip t
chains. In fish, exposure to MeHg has been shown to cause oxidative stress in brain of
cr
Atlantic cod (Gadus morhua) following intraperitoneal injection with MeHg (Berg et al., 2010) and in brain (Berntssen et al., 2003) and liver (Olsvik et al., 2011) of Atlantic salmon
us
(Salmo salar) after dietary exposure. Prolonged dietary exposure to MeHg has also been shown to affect growth, reproduction and neuronal function (Depew et al., 2012). Dietary
an
MeHg is highly bioavailable to fish and known to accumulate in all tissues (Berntssen et al., 2004; Clarkson, 1997), including the brain after traversing the blood-brain-barrier and also
M
muscle (Amlund et al., 2007; Berntssen et al., 2004). In contrast, elimination of MeHg from tissues is slow (Amlund et al., 2007) and as a consequence MeHg biomagnifies through
d
aquatic food chains (Fitzgerald and Mason, 1997; Morel et al., 1998). In aquaculture, the
te
inclusion of marine protein (fishmeal) in fish feed is the dominant source of mercury (and
Ac ce p
predominantly in the form of MeHg) for farmed fish. Hence, there is a considerable interest in identifying methods to be used in industry to decrease the bioaccumulation of MeHg in farmed fish to both reduce the risk of toxicity in fish and to minimise exposure to MeHg in humans.
Selenium is an essential cofactor for a number of selenoproteins, including glutathione peroxidases (GPxs) and thioredoxin reductase (TrxR), which have antioxidant functions (Arteel and Sies, 2001). Selenium is also a possible MeHg antagonist and has been shown to affect the kinetics of MeHg accumulation and tissue partitioning behaviour in fish (Bjerregaard et al., 1999; Bjerregaard et al., 2011; Branco et al., 2012; Dang and Wang, 2011; 4 Page 4 of 34
Deng et al., 2008; Huang et al., 2013; Ringdal and Julshamn, 1985; Sheline and SchmidtNielsen, 1977). However, these effects of selenium on MeHg uptake and elimination are complex and are dependent upon the chemical speciation of selenium. For example, dietary
ip t
selenite (SeO32-), selenocysteine (SeCys; HSeCH2CH(NH2)CO2H) and selenomethionine (SeMet; CH3SeCH2CH2CH(NH2)CO2H) increased the elimination of MeHg from goldfish
cr
(Carassius auratus) in a dose-dependent manner, whereas dietary selenate (SeO42-) had no effect (Bjerregaard et al., 2011). Dietary selenite has also been shown to increase the
us
elimination of MeHg from tissues, including muscle, liver and kidney, of rainbow trout (Oncorhynchus mykiss) (Bjerregaard et al., 1999) and dietary SeMet decreased the
an
accumulation of dietary MeHg in Sacramento splittail larvae (Pogonichthys macrolepidotus)
M
(Deng et al., 2008) and zebrafish (Danio rerio) (Penglase et al., 2014). Interestingly, coexposure to MeHg and SeMet in the diet increased the accumulation of selenium in splittail
d
larvae compared to larvae fed only SeMet, possibly indicating an increased requirement for
te
selenium in MeHg exposed fish (Deng et al., 2008). In contrast to these studies, dietary selenium (selenite, selenite, SeCys or SeMet) did not affect the kinetics of dietary MeHg in
Ac ce p
jarbua terapon (Terapon jurbua); the assimilation efficiency of MeHg was not reduced when the fish were either pre- or co-exposed to selenium (Dang and Wang, 2011). Collectively these studies indicate that dietary selenium may affect both accumulation and elimination of MeHg in fish, and vice versa, however the importance of selenium speciation, the interaction between selenium and MeHg, and the reasons for differences observed between species of fish are still poorly understood.
How selenium affects uptake, elimination and also toxicity of MeHg in fish is unclear. A possible mechanism is the formation of MeHg-selenol compounds; Selenium has been hypothesised to ameliorate MeHg toxicity through ligand exchange with MeHg-complexed 5 Page 5 of 34
thiol residues of proteins (Kahn and Wang, 2009). Conversely, the toxic effects of MeHg have been suggested to be due to a deficiency of selenium combined with an inhibition of selenoproteins as a result of MeHg-selenol complexation (Ralston and Raymond, 2010). For
ip t
example, the activities of GPx and TrxR were reduced in brain and liver of zebra-seabream (Diplodus cervinus) following waterborne exposure to MeHg (Branco et al., 2011; Branco et
cr
al., 2012). Nevertheless, in the digestive tract, the binding of MeHg to selenol groups could also lead to a decreased bioavailability of MeHg and reduced toxicity (Kahn and Wang,
an
us
2009).
The aim of the present study was to characterise the assimilation and depuration of dietary
M
MeHg, and to investigate the influence of dietary SeMet on MeHg bioaccumulation, in zebrafish. Zebrafish is an emerging model in toxicology and fish nutrition research, and have
te
Ac ce p
al., 2008; Liu et al., 2002).
d
been effectively applied to kinetic studies of dietary metals (Bjerregaard et al., 2011; Boyle et
2. Materials and methods
2.1 Experimental animals and study design The feeding trial was performed at the National Institute of Nutrition and Seafood Research, Bergen, Norway. The experiment was approved by the Norwegian Animal Research Authority and performed according to national and international ethical standards. During the pre-exposure acclimation period and during the experiments, fish were housed in a MultiRack System for zebrafish (Aquatic Habitats Inc., Apopka, FL, USA) with continuously aerated and triple-filtered (mechanical, chemical (activated carbon) and biological) 6 Page 6 of 34
recirculating water system (28.5°C, pH 7.5, 500 µS/cm 14h:10h light:dark photoperiod, and 10% daily water exchange). Zebrafish (males and females) were randomly distributed among 15 tanks (9L), in a triplicate
ip t
tank per treatment design and with 33-53 individuals in each tank, depending on the dietary treatment. The zebrafish were fed three times a day, at a total ration of 1.0% of their body
cr
weight daily. Zebrafish were fed the MeHg and/or selenium enriched diets, for eight weeks. A
us
control diet (no added mercury or selenium) was also included in the experimental design. After eight weeks of exposure two dietary groups (5 µg Hg/g diet; and 10 µg Hg/g and 5 µg
an
Se/g diet, respectively) were terminated. Groups fed a 5 µg Hg/g diet were transferred to the control diet, while groups fed the control and 10 µg Hg/g diets were split into six groups each.
M
Half of the groups were fed a control diet, while the other half were fed a 5 µg Se/g diet for four weeks. All groups of fish were kept in 3L tanks (11-15 fish per tank) during the four
Ac ce p
2.2 Experimental diets
te
d
weeks of depuration. The experimental design is summarised in Figure 1.
The experimental diets were produced by adding aqueous solutions of MeHg and/or SeMet to a commercial pelleted zebrafish diet (Aqua Schwarz, Göttingen, Germany). Methylmercury was added to diets as methylmercury-cysteine (MeHg-cys), which was prepared as described earlier (Glover et al., 2009). In brief, a stock solution of methylmercury(II) chloride (Alfa Aesar, Karlsruhe, Germany) was mixed with a stock solution of cysteine (dissolved in water; L-cysteine, Sigma-Aldrich, Seelze, Germany) in a 1:1.2 molar mixture. Selenium was added as SeMet (dissolved in water; Seleno-L-methionine, Sigma-Aldrich). The diets were produced in batches of 10 g with three ml of the appropriate solution (MeHg and/or SeMet) added gradually (1 ml at a time) to 10 g diet while stirring to avoid clumping of the pelleted diet.
7 Page 7 of 34
Following the addition of the solutions, the diets were dried at room temperature in a fume hood until a constant weight of 10 g was obtained (addition of solutions led to no net weight gain). The trays with batches of diet were occasionally shaken to avoid clumping of the
ip t
pelleted diet. The diets were stored at 4°C in the dark until required. Methylmercury alone was added to diets at nominal concentrations of 0, 5 and 10 µg Hg/g. Selenomethionine was
cr
added to one diet at a nominal concentration of 5 µg Se/g, and MeHg and SeMet were both added to one additional diet at nominal concentrations of 10 µg Hg/g and 5 µg Se/g,
us
respectively. The measured total mercury concentration of the diets were 0.08 ± 0.01 µg/g (n = 3) for the control diet, 5.2 ± 0.4 µg/g (n = 3) for the 5 µg Hg/g diet, 9.8 ± 0.5 µg/g (n = 3)
an
for the 10 µg Hg/g, 0.16 ± 0.04 µg/g for the 5 µg Se/g diet and 9.3 ± 0.6 µg/g (n = 3) for the
M
10 µg Hg/g + 5 µg Se/g diet. The measured selenium concentrations were 2.3 ± 0.1 µg/g (n = 3) for the control diet, 2.2 ± 0.5 µg/g (n = 3) for the 5 µg Hg/g diet, 2.3 ± 0.1 µg/g (n = 3) for
d
the 10 µg Hg/g, 4.3 ± 0.1 µg/g for the 5 µg Se/g diet and 4.3 ± 0.2 µg/g (n = 3) for the 10 µg
te
Hg/g + 5 µg Se/g diet. All diets were balanced with cysteine and/or methionine (L-
Ac ce p
methionine, Sigma-Aldrich) to equal the nominal levels in the 10 µg Hg/g + 5 µg Se/g diet.
2.3 Tissue samplings
To guarantee sufficient tissue for measurements of mercury and selenium, tissues (muscle, liver and brain) of n = 3 zebrafish from each tank were pooled. Zebrafish (n = 3 per tank) were sampled at weeks 2 and 8 during the exposure period and at the end depuration period (Week 12). In addition zebrafish were sampled at the start of the feeding trial (n = 3, replicate samples). The zebrafish were sampled randomly and immediately killed by an overdose of MS-222 (0.5 g/L; ethyl 3-aminobenzoate methanesulfonate; Sigma-Aldrich). Length and weight of the zebrafish were recorded and brain, liver and muscle were excised. Pooled samples (n = 3 fish per pool) were kept on ice during sampling, and then stored at -20 °C until 8 Page 8 of 34
further analyses. For some zebrafish and time points only muscle was analysed as muscle is an important site of accumulation of mercury (Berntssen et al., 2004) and of importance
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within aquaculture.
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2.4 Mercury and selenium determination by inductively coupled plasma mass spectrometry
Total mercury and selenium were determined in diets and samples by inductively coupled
us
plasma mass spectrometry (ICPMS) after microwave assisted decomposition as previously described (Julshamn et al., 2007). In brief, the samples were digested in 65% nitric acid (2 ml;
an
Suprapur, Merck, Darmstadt, Germany) and 30% hydrogen peroxide (0.5 ml; Merck) using a microwave digestion system (Ethos 1600; Milestone, Sorisole, Italy). The solutions were
M
diluted to 10 ml with deionised water (>17 MΩ/cm; Nanopure System; Barnstead, Dubuque, IA, USA). Total mercury and selenium concentrations in all samples were determined by
d
ICPMS (Agilent ICPMS 7500c; Yokogawa analytical systems, Tokyo, Japan) equipped with
te
an autosampler (ASX-500; CETAC Technologies, Omaha, NE, USA). Data were collected
Ac ce p
and processed using the Agilent Chemstation ICPMS software (Agilent Technologies, Palo Alto, CA, USA). Rhodium was used as an internal standard to correct for any drift of the instrument. The accuracy of the analytical method was assessed by analysis of two certified reference materials; lobster hepatopancreas (TORT-2; National Research Council Canada, Ottawa, Ontario, Canada; for mercury; certified value 0.27 ± 0.06 µg Hg/g; obtained value 0.27 ± 0.00 µg Hg/g, n = 2; for selenium; certified value 5.63 ± 0.67 µg Se/g; obtained value 4.91 ± 0.18 µg Se/g, n = 2) and oyster tissue (SRM 1566b; National Institute of Standards and Technology, Gaithersburg, MD, USA; for mercury; certified value 0.0371 ± 0.0013 µg Hg/g; obtained value 0.032 ± 0.001 µg Hg/g, n = 4; for selenium; certified value 2.06 ± 0.15 µg Se/g; obtained value 2.05 ± 0.06 µg Se/g, n = 4). The limit of quantification (LOQ) of the method is 0.005 and 0.01 µg/g for mercury and selenium, respectively. 9 Page 9 of 34
2.5 Statistical analyses The data presented were evaluated statistically using Statistica® (Ver. 10; StatSoft, Tulsa,
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OK, USA). All data sets were first tested for normality (Shapiro-Wilk test) and homogeneity
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of variances (Levene’s test). To detect significant differences between treatment groups, data sets of fish weight, and mercury and selenium concentrations in muscle, liver and brain were
us
analysed using one-way analysis of variance (ANOVA) with post-hoc Tukey’s honestly significance test. For all analyses a 95% confidence interval was applied with a level of p
