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In vivo Mercury Demethylation in Marine Fish Xun Wang, Fengchang Wu, and Wen-Xiong Wang Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 18 May 2017 Downloaded from http://pubs.acs.org on May 18, 2017
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In vivo Mercury Demethylation in a Marine Fish (Acanthopagrus schlegeli)
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Xun Wang†, Fengchang Wu§, Wen-Xiong Wang*,†
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† Division of Life Science, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong, and Marine Environmental Laboratory, HKUST Shenzhen Research Institute, Shenzhen 518057, China § State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China
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*
Corresponding author: wwang@ust.hk phone: (852) 23587346
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ABSTRACT Mercury (Hg) in fish has attracted public attention for decades, and
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methylmercury (MeHg) is the predominant form in fish. However, the in vivo MeHg
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demethylation and its influence on Hg level in fish have not been well addressed. The
22
present study investigated the in vivo demethylation process in a marine fish (black
23
seabream, Acanthopagrus schlegeli) under dietary MeHg exposure and depuration,
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and quantified the biotransformation and inter-organ transportation of MeHg by
25
developing a physiologically based pharmacokinetic (PBPK) model. After exposure,
26
we observed a 2-fold increase of the whole-body inorganic Hg (IHg), indicating the
27
existence of in vivo demethylation process. The results strongly suggested that the
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intestine played a predominant role in MeHg demethylation with a significant rate
29
(6.6±1.7 d-1) during exposure, whereas the hepatic demethylation appeared to be an
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extremely slow (0.011±0.001 d-1) process and could hardly affect whole fish Hg level.
31
Moreover, demethylation in the intestine served as an important pathway for MeHg
32
detoxification. Our study also pointed out that in vivo MeHg demethylation could
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influence Hg level and speciation in fish although food is the major pathway for Hg
34
accumulation. Enhancing in vivo MeHg biotransformation (especially in the intestine)
35
could be a potential key solution in minimizing Hg contamination in fish. The related
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factors involved in intestinal demethylation deserve more attention in the future.
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Keywords: Methylmercury; in vivo demethylation; marine fish; PBPK modeling.
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TOC art
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INTRODUCTION
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Mercury (Hg) is a global and highly toxic metal pollutant attracting the world’s
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attention.1,2 As one of the few metals known to biomagnify along the food chains in
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aquatic environments, Hg [especially methylmercury (MeHg)] can be easily
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accumulated and concentrated by fish.3 The elevated levels of MeHg in fish have
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raised public concern on fish consumption.4,5 It is intriguing that the majority of Hg in
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fish is presented in methylated form,6,7 although inorganic Hg (IHg) is the
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predominant form (>95%) in natural water.8 Traditionally, the high levels of MeHg in
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fish were considered to be derived from trophic transfer9 and attributed to its higher
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biomagnification potential than IHg.10,11 Indeed, the in vivo MeHg biotransformation
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(demethylation) can be a potential key process that determines the final biological fate
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and speciation of Hg in fish.12 Since MeHg could be converted into IHg through
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demethylation, the occurrence and rate of this reaction would directly affect the
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relative abundance of IHg versus MeHg in fish. However, this process has not been
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thoroughly investigated and still remains unclear.
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In the aquatic environment, demethylation can take place via physical
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(photodemethylation),13 chemical [selenium mediated]14 and biological processes
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(microbial activities).15 However, in vivo demethylation in fish has not been well
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described. Joiris and Holsbeek16 observed that MeHg ratio in the liver of two sardines
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decreased with age (from 50% to 20%), suggesting that it might reflect the existence
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of a slow demethylation process. Based on the quantifications by Hg stable isotopes,
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however, Wang et al.17 suggested that the decreased MeHg ratio in the liver was likely
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due to MeHg inter-organ transportation from liver to muscle rather than
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demethylation. Feng et al.18 observed that demethylation occurred in zebrafish (Danio
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rerio) but could not distinguish the specific organ for this process. It is also debatable
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where the in vivo MeHg biotransformation occurs. As a detoxification organ, the liver
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is naturally suspected to be the major site for demethylation, but contradictory results
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were found in previous studies. Gonzalez et al.19 observed that MeHg represented 66%
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of mercury in the liver of zebrafish (D. rerio) at day 0 and decreased to 36% after 63
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days of MeHg exposure, suggesting that a demethylation process was in place.
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However, for the same fish species, no demethylation process was observed in the
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liver during 62 days exposure to dietary MeHg.20 Another possible site for MeHg
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biotransformation is the digestive tract, which not only serves as the first barrier for 4
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the ingested Hg, but may also participate in the transformation process.21 Significant
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demethylation was observed to occur in the gut lumen of MeHg-treated rat and
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intestinal flora was considered to be responsible for this process.22 Moreover,
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Rowland et al.23 suggested that the IHg derived from MeHg demethylation in the gut
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of mice did not re-enter the general circulation. Given that food is the predominant
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route for Hg exposure to fish, intestinal demethylation might be a potentially
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important process affecting the uptake and accumulation of Hg by fish. However,
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there is no abundant evidence of the presence of intestinal demethylation in fish, and
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its potential influence on whole-body Hg burden has never been considered.
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Therefore, it is necessary to find out whether, where and how fast the MeHg
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biotransformation occurs so as to better understand the internal handling of MeHg by
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fish.
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Physiologically based pharmacokinetic (PBPK) modeling is a useful tool for
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simulating the accumulation, transformation and elimination of toxic compounds
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among multiple tissues of organisms.24 Taking into account the effects of exposure
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scenarios, routes, doses, and species, PBPK modeling can not only illuminate the
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disposition of compounds in organisms, but can also evaluate the relative importance
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of different tissues to specific physiological-biochemical processes.25 PBPK modeling
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has been successfully utilized to describe the distribution and elimination of Hg in
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fish and has shown its power in elucidating the roles of different tissues in the internal
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handling of Hg.26,27 However, this mathematical tool has never been applied for
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studying the in vivo MeHg biotransformation in fish. In this study, we investigated the
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dynamic changes of MeHg and IHg in five different compartments of black seabream
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(Acanthopagrus schlegeli) under dietary exposure to MeHg and depuration, and
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constructed a PBPK model to simulate the disposition of MeHg and IHg in these
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compartments. Based on the direct observations and simulation results, the present
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study was aimed to (1) explore the existence or not of the in vivo MeHg
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demethylation; (2) distinguish the possible site(s) for demethylation; (3) evaluate the
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influence of demethylation on Hg level and speciation of fish. The determined
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kinetics by our modeling could help to support the observations from the perspective
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of mathematics.
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METHODS AND MATERIALS 5
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Fish Collection and Food Preparation. Black seabream (Acanthopagrus schlegeli)
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is widely distributed in coastal environment and it is an excellent species for culture
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due to its tolerance to a wide range of environmental conditions.28 Fish in similar size
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(length 10 cm, fresh weight 15 g) were collected in Sai Kung, Hong Kong, and
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transferred into the sand-filtered seawater at 25 oC with a 14:10 h light: dark cycle.
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The fish were acclimated for 2 weeks by feeding with clean food pellets (New Life
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International, Inc.).
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The measured concentrations of total mercury (THg) and methylmercury (MeHg)
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in clean fish diet were 0.035±0.002 µg g-1 dw (dry weight) and 0.022±0.005 µg g-1 dw,
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respectively. The concentration of MeHg in the spiked food was set as 1.0 µg g-1 dw,
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which was representative for MeHg in realistic prey for black seabream (e.g.
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mollusks).29,30 The spiked fish diet was prepared by incubating 100 g of clean
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commercial fish food with 125 mL of freshly prepared solution (100 µg MeHg added
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as MeHgCl). Then the food pellets were dried at room temperature for 2 days. The
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measured concentrations of THg and MeHg in the spiked fish diet were 1.07±0.10 µg
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g-1 dw and 1.03±0.11 µg g-1 dw, respectively.
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Dietary Exposure, Depuration and Sampling. After acclimation in the laboratory,
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fish were randomly selected and divided into two groups (control and MeHg-exposed
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group). Four aquariums (size of 60 × 30 × 45 cm3) were used for each group, with 20
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fish in each aquarium for MeHg-exposed group and 4 fish in each aquarium for the
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control group. The fish in the MeHg-exposed group were fed the MeHg spiked food
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pellets, whereas the control group was fed clean diet during the exposure period. The
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exposure lasted for 12 days and feeding was carried out twice a day at a rate of 0.016
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g dry weight g-1 wet weight d-1. The diet consumption time lasted for 1 h and the
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feeding behavior was monitored to ensure that almost all food pellets were eaten
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(>95%). Then the uneaten food pellets and feces were siphoned off. After the
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exposure, the fish were depurated for another 30 days. Fish in both groups were fed
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clean food pellets at the same rate. During the entire experiment period, fish were kept
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under the same conditions as those during the acclimation period, and the seawater
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was cycled at a flow rate of 3 L/min to ensure that the water was clean.
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The MeHg-exposed fish were sampled at 0, 3, 6, 9, 12, 13, 15, 17, 20, 24, 28, 32,
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37 and 42 d, whereas sampling of the control group took place every 6 days. Each
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aquarium was considered to be one replicate for each treatment, and one fish was 6
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randomly collected from each aquarium at each sampling time point. Then fish were
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rinsed by deionized water and narcotized in cold ice water. The caudal fin was cut off
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and the drained blood was collected by capillary pipet. Fish were then dissected and
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separated into intestine, gills, liver and carcass. After the weighing, the fish samples
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were freeze-dried and stored for further measurements.
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Chemical and Statistical Analysis. All the fish samples as well as fish diet (clean
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and spiked) were determined for THg and MeHg concentrations. The analysis of THg
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followed the method of EPA 7474 with a few modifications. Briefly, the homogenized
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samples (0.05-0.1 g dw) were digested at 80 oC with 2 mL of aqua regia in a heating
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block for 12 h. The digested solution was diluted as appropriate. An aliquot of the
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diluted sample was added into the mixture of hydrochloride/bromate/bromide to
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ensure that all forms of Hg were oxidized into Hg(II) ions. Before analysis, samples
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were reduced by addition of sodium chloride hydroxylamine hydrochloride. THg was
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then measured by cold vapor atomic fluorescence spectrometry (CVAFS, QuickTrace
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8000, USA, detection limit < 0.1 ng L-1). MeHg analysis followed the method of EPA
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1630.31 Approximately 0.05 g of tissues was digested at 80 oC with 2 mL of 25%
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KOH/methanol solution in an oven for 4 h. The extract was diluted and 20-100 µL of
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the solution was buffered with sodium acetate at pH 4.9, and ethylated by freshly
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thawed 1% NaBEt4 solution. MeHg was measured by an automated MeHg analytical
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system (MERX, Brooks Rand, USA, detection limit < 0.002 ng L-1). To validate the
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accuracy of elemental determinations, standard reference materials (Fish protein
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DORM-4, National Research Council of Canada) was concurrently digested and
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analyzed for every batch of 20 samples. The recovery rates were 93-105% for THg
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and 90-106% for MeHg. In this study, the concentrations of inorganic Hg (IHg) in
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specific organ were calculated by subtracting MeHg concentrations from THg
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concentrations. Results are reported in ng Hg g-1 for THg, MeHg and IHg on a fresh
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weight (F.W) basis.
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Statistical Analysis. Comparisons of THg, MeHg and IHg concentrations between
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time points were performed using one-way analysis of variance (ANOVA) followed
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by Duncan test. Comparisons were considered statistically significant at p < 0.05 and
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statistical analyses were performed in SPSS 17.0. The F-value was used to indicate
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the extent of differences in THg, MeHg and IHg concentrations between time points.
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Model Development. 7
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Basic definitions. A pharmacokinetic model was constructed to describe the uptake,
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distribution, transformation and elimination of MeHg in black seabream, as shown in
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Fig. 1. In this study, each chemical form of Hg (MeHg and IHg) in each organ was
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treated as an independent compartment so that the kinetics of each compartment could
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be determined. A compartment is a theoretical construct that may include several
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different organs or tissues (e.g. carcass) or could be one part of a specific organ (e.g.
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gut wall). The modeling can be viewed as a hypothesis to be tested on the
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experimental data, and the structure of the model is then altered until the fitting on the
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data is satisfactory. The blood could be considered as a “carrier” that distributed Hg to
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other organs. The intestine was not only an important site for uptake and elimination
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for Hg, but was also suggested to take a significant part in the generation of IHg in
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MeHg-treated animals.22 Besides that, the liver was assumed to be another site for
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MeHg demethylation, as suggested by previous studies.19,32 The gill was chosen
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owning to its great contribution in IHg excretion to outside environments.26 The
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carcass, accounting for >90% of body weight, was regarded as the largest pool for
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Hg.27
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Assuming that the transport between the compartments followed first-order kinetics, it could be expressed by the following equation: Flux(i, j) = k(i, j)•Qj
(1)
-1
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where Flux(i, j) refers to the mass flux (ng d ) of Hg (MeHg or IHg) from the jth to the
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ith compartment and k(i, j) (d-1) is the rate coefficient between the compartments. Qj is
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the total amount of Hg (ng) in the jth compartment at time t, and equals to the Hg
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concentration in the jth compartment (Cj) multiplied by its fresh weight (wj). The Hg
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content in the blood was calculated by taking the total blood volume of the fish into
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account, assuming 60 ml blood/kg tissue in teleosts.33 The fish weights parameters
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and model equations are listed in Table S1 and Table S2, respectively.
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Simulation of absorption, transformation and elimination of MeHg in the gut
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lumen. Given that fish were fed twice a day, the intestine could not be completely
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empty and some of the food in digestion (or feces) would be inevitably retained in the
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gut lumen when the fish were sampled. In this case, the determined Hg concentrations
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of the intestine in our study should be comprised of those in the feces and the gut
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tissue. To simulate the physiological process (uptake and elimination) and
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biotransformation that MeHg involved in the digestive tract, the intestine 8
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compartment was divided into two independent sub-compartments: Chyme and gut
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wall.34 Chyme is the semifluid mass of food mixed with digestive solution and can be
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considered as the “precursor” of feces. MeHg in the chyme could be absorbed by the
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gut tissue (Flux(2,7)) or demethylated into IHg (Flux(17,7)), some of which could also be
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taken up (Flux(12,17)). The residual MeHg and IHg in the chyme were finally
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eliminated to outside through the feces (Flux(10,7) and Flux(10,17)). Concentrations of
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Hg in the intestine can be expressed by the following equation:
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Cintestine = (Qchyme + Qgut wall)/(wchyme + wgut wall)
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where Cintestine refers to the determined value of MeHg or IHg for the intestine,
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whereas Qchyme and Qgut wall represent the total amount of MeHg or IHg in the chyme
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and the gut wall at time t. It should be noted that Qchyme and Qgut wall were fitted by the
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modeling. wgut wall refers to the fresh weight of the empty intestine and was obtained
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from the starved fish in the preliminary experiment. For the convenience in simulation,
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wchyme was assumed to be a constant and equaled to be the weight of fed food in one
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meal.
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(2)
Simulation of IHg disposition in the liver. As a highly perfused organ responsible
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for detoxification, the liver has been shown to play a fundamental role in
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redistribution of MeHg and IHg in fish.35 To simulate the IHg behaviors in the liver,
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we divided it into two sub-compartments: the storage pool and the active pool (Fig. 1).
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The former one represented the IHg derived from hepatic demethylation (Flux(20,4))
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and which could not be transferred out from the liver, since it has been suggested that
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the formed IHg from demethylation was sequestered in the liver of fish.32,36 The latter
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one referred to the active IHg that could be exchanged with blood (Flux(14,11) and
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Flux(11,14)). Concentrations of IHg in the liver can be expressed by the following
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equation: Cliver-IHg = (Qstorage-IHg + Qactive-IHg)/ wliver
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(3)
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where Cliver-IHg refers to the determined value of IHg for the liver, whereas Qstorage-IHg
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and Qactive-IHg represent the simulated mass (ng) of IHg in the storage pool and active
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pool at time t. wliver refers to the fresh weight of the liver. It should be noted that the
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“storage” and “active” pools were man-made mathematical concepts, which were
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only used to represent the amount of IHg to be stored in the liver or exchanged with
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blood.
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Data fitting. We used the SAAM II modeling software version 2.3.1 (SAAM 9
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Institute, University of Washington, Seattle, WA, USA) to construct the modeling and
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calculate the kinetic parameters. SAAM II has been successfully used to simulate the
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distribution and elimination of Hg as well as other trace metals within fish body.27,37
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The modeling structure was constructed based on two guiding principles: it should
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contain the fewest compartments to adequately describe the data; it should reflect the
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realistic physiology or metabolic process. Thus the final modeling structure was
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reached by a process of trial. During the fitting process, the parameters were given
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initial values comparable to published data on MeHg and IHg distribution in fish, and
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were allowed to vary until the best fitting was reached. The software would finally
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give the mean and the standard deviation (SD) of the parameters. To provide the best
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fit, the software could iteratively minimize an objective function based on the Akaike
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information criterion (AIC) and the Schwarz–Bayesian information criterion (BIC).
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These two criteria are a function of the goodness of fit, the number of adjustable
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parameters, and the total number of data points. When comparing two potential
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modeling structures, lower values of AIC and BIC indicated the modeling better
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described the data with the least number of parameters.37 The quality of parameter
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estimates were evaluated based on SDs of the parameters, the parameter correlation
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matrixes and the appearance of the data-model plots. If the RSD (the ratio of the
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standard deviation to the mean value) was lower than 0.5, the parameter would be
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considered to be different from zero with 95% confidence. If the correlation
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coefficient between two parameters was higher than 0.9, it would indicate that these
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two parameters function in a similar manner in the fitting, and the modeling would be
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overparametrized.38
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RESULTS AND DISCUSSION
In vivo demethylation of MeHg. In this study, the concentrations of THg and
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MeHg in the control group had no significant differences from the initial to the end of
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the experiment (Fig. S1), indicating that the effects of clean food pellets and seawater
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on fish were negligible. Concentrations of total mercury (THg), methylmercury
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(MeHg) and inorganic mercury (IHg) in the five compartments (blood, intestine, gills,
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liver and carcass) of Acanthopagrus schlegeli are shown in Fig. 2. During the
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exposure period (0-12 d), both THg and MeHg concentrations in all the five
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compartments increased significantly, whereas they showed different trends during 10
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the depuration period (12-42 d) (Figs. 2 a-d). For blood, gills and intestine, THg and
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MeHg decreased sharply during the first 8 days of depuration (12-20 d) and were then
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stable until the end of experiment. For carcass, THg declined slightly but MeHg
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showed no obvious change. Moreover, THg in the liver decreased dramatically until
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the 20th day (F = 25.6) and kept stable from then on, whereas liver MeHg kept
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declining during the depuration period (F = 32.0). As shown in Figs. 2 e and f, IHg
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concentrations in the blood, intestine, gills and carcass increased significantly during
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exposure, then decreased continuously during depuration and reached to the same
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level as the initial values at the end of experiment. However, IHg in the liver showed
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an opposite trend, which declined sharply (F = 21.2) and reached to the bottom at the
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end of exposure, then increased greatly (F = 29.6) during the last 30 days. At the end
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of experiment, both THg and MeHg concentrations followed this trend: carcass >
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liver >> gills ~ intestine > blood, whereas IHg concentrations followed this trend:
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liver >> intestine ~ gills > carcass > blood. The estimated parameters of MeHg and
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IHg for different compartments are listed in Tables 1 and 2, respectively. The
287
simulated curves for MeHg and IHg in different compartments of MeHg-exposed fish
288
are shown in Figs. 3 and 4, respectively. Overall, the model-data plots were well fitted.
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The RSDs of most estimated parameters were lower than 0.5, indicating sufficient
290
statistical accuracy. Besides, all of the correlation coefficients between parameters
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were lower than 0.9, suggesting that all the parameters functioned independently and
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the modeling was not overparameterized.
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The whole-body concentrations were calculated by adding up the products of the
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concentrations of each organ multiplied by its proportion of whole-body weight. As
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shown in Fig. 2b, MeHg concentrations in whole fish increased greatly from 41 to 214
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ng Hg g-1 F.W (fresh weight) during the exposure period, indicating that MeHg could
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be easily absorbed and accumulated in fish. More intriguingly, whole-body IHg
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increased by 2-fold (from 21 to 42 ng Hg g-1 F.W) during this period and the newly
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accumulated IHg (21 ng Hg g-1 F.W) accounted for a considerable proportion (> 10%)
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of THg (193 ng Hg g-1 F.W) (Fig. 2f), demonstrating that IHg was also deposited in
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fish. Given that MeHg was the only significant source for Hg intake (THg and MeHg
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in the spiked fish diet were 1.07±0.10 µg g-1 dw and 1.03±0.11 µg g-1 dw,
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respectively), our study strongly suggested that demethylation of MeHg occurred in
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black seabream. These observations can also be supported by modeling results. The 11
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MeHg uptake, demethylation and elimination rates during exposure were estimated to
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be 180, 50 and 10 ng Hg d-1 (on average), respectively (Fig. 5). This suggested that
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more than 20% of the total ingested MeHg was demethylated into IHg, which
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subsequently significantly affected Hg composition in fish. Thus, the modeling results
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pointed out that demethylation was a significant process and played an important role
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in MeHg disposition. Chumchal et al.36 observed that MeHg comprised the majority
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of THg in the muscle of spotted gar (Lepisosteus oculatus), whereas IHg was the
312
predominant form in the liver, suggesting that demethylation occurred in this fish
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species. However, Drevnick et al.39 ascribed the low MeHg and high IHg ratios in the
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liver of northern pike (Esox lucius) to the IHg uptake from dietary source but not
315
demethylation. Eagles-Smith et al.40 also observed significant taxonomic differences
316
in demethylation ability in water birds. These observations suggested that the
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demethylation potential in fish and other vertebrates might be species specific. Thus,
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there is a further need to investigate the occurrence of demethylation in other marine
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fish species. Overall, based on the direct observations and mathematical modeling, we
320
provided direct evidences on the existence of in vivo MeHg demethylation and
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revealed its importance in the internal handling of MeHg by the marine fish black
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seabream.
323
During the depuration period, whole-body concentrations of IHg decreased
324
significantly to 19 ng Hg g-1 F.W (the same level to the beginning) when depuration
325
was ended (Fig. 2f). This suggested that the IHg derived from MeHg demethylation
326
could be efficiently eliminated. The elimination rate constant (ke, d-1) for IHg can be
327
calculated as the absolute value of the slope of linear regression of the natural log of
328
the percentage of IHg retained in whole body against depuration time.41 The estimated
329
ke value for IHg was 0.024±0.002 d-1, which was comparable to that measured in the
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same fish species (0.031 d-1for 15 g F.W fish).11 However, MeHg in whole fish
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showed no significant change during the depuration period (Fig. 2d), indicating that
332
MeHg was difficult to be eliminated and its loss was negligible within the timeframe
333
of our study. Traditionally, food chain transfer is considered to be the predominant
334
pathway for Hg accumulation3 and the high proportion of MeHg in fish is ascribed to
335
its higher biomagnification potential than IHg.10,11 However, our study showed that
336
there could be a considerable amount of IHg generated from demethylation and
337
accumulated by fish even MeHg was the only Hg source. The final high MeHg ratio (> 12
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90%) in whole fish (Fig. S2) was caused by the relatively fast elimination of IHg and
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extremely slow loss of MeHg. Therefore, our study suggested that the Hg deposited in
340
fish could be derived from varied sources (such as demethylation) rather than from
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food only. The extremely high MeHg proportion observed in wild fish7,42 could be
342
attributed to a series of complicated physiological-biochemical processes, in which
343
the in vivo MeHg biotransformation could make great influence on Hg composition in
344
fish. Given that significant amount of MeHg was transformed into IHg and could be
345
eliminated within rather short period, demethylation helped to reduce the
346
accumulation of MeHg and diminish its toxic effects on fish. It is considered that fish
347
tend to store the largest amount of MeHg in the muscle, thus protecting other tissues
348
from MeHg toxicity.12 However, this study suggested that demethylation could be
349
another pathway for MeHg detoxification in fish, and enabled a better understanding
350
of the detoxification and elimination of MeHg by fish.
351 352
Demethylation sites in fish. Demethylation of MeHg was traditionally suspected
353
to occur mainly in the liver of fish.16,19 However, based on the following two reasons,
354
our study strongly suggested that the intestine was the major site for demethylation
355
when fish were exposed to MeHg. Firstly, the 2-fold increase in whole-body IHg
356
concentration indicated that a large amount of IHg was deposited in fish. If
357
demethylation primarily took place in the liver, there should be a significant amount
358
of IHg produced in the liver and its IHg level should be greatly elevated. However,
359
liver IHg declined significantly (from 89 to 37 ng Hg g-1 F.W) during exposure (Fig.
360
2f), suggesting that the contribution of hepatic demethylation to IHg accumulation in
361
whole fish should be rather limited. On the contrary, IHg in the intestine increased
362
greatly (from 27 to 138 ng Hg g-1 F.W) during exposure (Fig. 2f) and possessed
363
around 40% of THg at the end of exposure (Fig. S2b), suggesting that significant
364
amount of IHg was generated in the gut lumen. Since MeHg was orally taken by fish
365
in this study, the intestinal flora might play an important role in MeHg
366
biotransformation.23 Secondly, the simulated demethylation rate in the intestine was
367
around 50 ng d-1 (on average) during exposure, whereas that in the liver was only
368
around 1 ng d-1 (Fig. 6). This strongly demonstrated that the intestine rather than liver
369
dominated in MeHg demethylation when fish were under MeHg exposure. Previously,
370
Feng et al.18 observed an important contribution (~35%) of IHg in the feces resulting 13
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from MeHg demethylation in zebrafish (Danio rerio), but could not distinguish the
372
demethylation site(s) due to the influence by multiple transport steps. Here we
373
synthetically considered the processes of demethylation and inter-organs
374
transportation, and evaluated the contributions of the liver and intestine in
375
demethylation by utilizing PBPK modeling. The results suggested that the intestine
376
was not only the major site for demethylation, but might also play an important role in
377
the regulation of Hg level and detoxification of MeHg. Given that significant amount
378
of MeHg was demethylated in the intestine, the decreased MeHg uptake could protect
379
other tissues from its toxicity. The generated IHg from demethylation could be
380
subsequently absorbed, thus greatly affecting Hg composition in fish. However, the
381
related gut microflora and the possible mechanism involved in intestinal
382
demethylation remains unclear. One possible explanation is that the elevated level of
383
MeHg in the gut lumen might induce the expression of genes encoding
384
organomercurial lyase (MerB) and mercuric reductase (MerA),43 thus leading to
385
higher demethylation rates. Some specific strains of anaerobic (e.g. iron-reducing
386
bacteria)44 and aerobic microbes15 could take part in the demethylation process as they
387
might be present in fish digestive tracts.
388
During depuration, IHg concentrations in the intestine declined greatly (Fig. 2f).
389
Since fish were fed with clean food within this period, there was no MeHg that could
390
be utilized by intestinal microflora. Thus, intestinal demethylation was stopped and
391
the liver was the major site for demethylation within this period. It is notable that
392
MeHg concentrations in the liver declined significantly (from 177 to 110 ng Hg g-1
393
F.W) from Day 20 to Day 42, whereas IHg concentrations increased from 48 to 108
394
ng Hg g-1 F.W (Figs. 2d and 2f). More intriguingly, THg concentrations in the liver
395
were kept stable within this period (Fig. 2b). All these observations suggested that the
396
IHg derived from demethylation was immobilized in the liver and could not be
397
transferred out. Perrot et al.45 also observed that MeHg was demethylated in vivo and
398
the formed IHg was stored in the liver of mammals. In fish, IHg was found to be
399
co-localized with selenium (Se) in the liver and a positive correlation between their
400
concentrations was observed,32,46 suggesting that Se might be involved in the
401
demethylation process in the liver. The possible mechanism in hepatic demethylation
402
could be via the formation of HgSe(s),47,48 which is inert and sequestered within
403
hepatic cells.49 However, it should be noted that liver IHg greatly decreased during the 14
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404
first 9 days, suggesting that the original IHg in the liver was “active” and could be
405
transferred out. Since the newly accumulated MeHg tended to be firstly transferred to
406
the liver, the “active” IHg could be replaced by MeHg and distributed to other parts of
407
fish. To simulate the different behaviors of IHg in the liver, we divided liver IHg into
408
two subcompartments: active pool and storage pool. The former one refers to IHg that
409
can be exchanged with blood and the latter one represented IHg derived from
410
demethylation and stored in the liver. The simulated IHg mass in the active pool of
411
liver declined in a higher rate than IHg production from demethylation during the
412
initial period (Fig. S3). Thus the modeling successfully described the IHg kinetics in
413
the liver and explained why IHg decreased during the first 9 days and then gradually
414
increased till the end. Given that hepatic demethylation was a rather slow process
415
(estimated rate constant equaled to 0.011±0.001 d-1) and the formed IHg could not
416
participate in body circulation, its influence on Hg deposition in whole fish was
417
limited in this study.
418
In this study, the methylation process was not considered for the following two
419
reasons. Firstly, the fish were fed with MeHg only, thus the substrate for the
420
methylation process was not available. Another possibility is that the generated IHg in
421
the intestine lumen might be transformed back into MeHg. However, Lu et al.44
422
observed that both methylation and demethylation could be carried out by the same
423
anaerobic bacteria, thus the direction of reaction depends on which species of Hg is
424
mainly provided to the bacteria. Since fish were fed with MeHg-spiked food only, the
425
methylation could hardly occur in the intestine. Secondly, the methylation of IHg into
426
MeHg in fish is an extremely slow process. Wang et al.17 found that only 0.67-1.60%
427
of the ingested IHg was methylated into MeHg in freshwater fish during two-month
428
depuration. Given that the depuration in our study lasted for one month, no more than
429
1% of the generated IHg could be converted back into MeHg. Thus, the influence of
430
methylation on the disposition of IHg and MeHg within fish body was negligible in
431
our study. Its contribution needs to be further investigated within a longer time scale
432
or with an elevated IHg level in fish diet.
433 434
Implication on MeHg control in fish. Hg (especially MeHg) in fish, as the most
435
important route for humans exposed to Hg, has raised particular concern to public
436
health for decades.4,50 Decreasing the bioavailability of MeHg has been long 15
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437
considered to be the major pathway to reduce the MeHg accumulation by fish.12
438
However, our study found that Hg level and speciation in fish could be greatly
439
affected by in vivo MeHg biotransformation. If the demethylation process could be
440
enhanced within fish body, less MeHg would be accumulated by fish. Compared to
441
the liver, the intestinal demethylation possessed higher potential to be implicated on
442
MeHg control in fish for the following two reasons. Firstly, the intestine (rather than
443
the liver) played a dominant role in MeHg demethylation when fish were exposed to
444
MeHg. Given that the intestine was also the major site for MeHg uptake, intestinal
445
demethylation could decrease the amount of MeHg assimilated by fish, thus helping
446
to control the MeHg accumulation from the source. Besides that, there is no need to
447
concern about the extra IHg uptake derived from intestinal demethylation, since IHg
448
could be eliminated within short period. Secondly, the influence of hepatic
449
demethylation on whole-fish Hg level was negligible due to its extremely low rate.
450
For fish, more than 80% of the MeHg body burden is stored in muscle.12 However,
451
the MeHg elimination from muscle is extremely slow attributing to its tight binding
452
with cysteine-rich proteins.51 Since the MeHg transfer from muscle to liver was rather
453
limited, demethylation in the liver could hardly reduce MeHg accumulation by fish.
454
Overall, our study suggested that enhancing intestinal demethylation could be a
455
potentially useful pathway for MeHg control in fish. The factors that may influence
456
this process (including the specific bacteria strains, temperature, pH, etc.) deserve
457
more investigations in the future.
458
Our study for the first time provided direct evidences on the existence of in vivo
459
MeHg demethylation in a marine fish (Acanthopagrus schlegeli) and quantified the
460
biotransformation and inter-organs transfer processes of MeHg by utilizing PBPK
461
modeling. Based on the observations and simulation results, the present study strongly
462
suggested that the intestine played the dominant role in demethylation under MeHg
463
exposure and intestinal demethylation occurred in a significant rate. Moreover,
464
demethylation in the intestine served as an important pathway for MeHg
465
detoxification. However, hepatic demethylation was an extremely slow process and
466
contributed very little to whole-body Hg level and speciation. Our study also pointed
467
out that in vivo MeHg demethylation could influence Hg level and speciation in fish
468
although diet is the major pathway for Hg accumulation. Enhancing in vivo MeHg
469
biotransformation (especially in the intestine) is suggested to be a potential key 16
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solution in minimizing Hg contamination in fish. The related factors involved in
471
intestinal demethylation are needed to be further investigated in the future.
472 473
ASSOCIATED CONTENT
474
Supporting Information
475
Figure S1: Concentrations of THg (a, b) and MeHg (c, d) in different compartments
476
(blood, intestine, gill, liver and carcass) and whole body of Acanthopagrus schlegeli
477
fed with clean fish diet. Data are means ± SD (n = 4). Figure S2: MeHg and IHg ratio
478
(percentage of THg) in different compartments (blood, intestine, gill, liver and carcass)
479
and whole body of Acanthopagrus schlegeli during exposure and depuration; Figure
480
S3: The simulated IHg mass (ng) in the active pool (black curve) and storage pool
481
(red curve) of the liver in Acanthopagrus schlegeli during exposure and depuration;
482
Table S1: Fish weights parameters used for calibration; Table S2: Equations used for
483
calibrations. Table S3: Analysis of variance of THg, MeHg and IHg concentrations in
484
five compartments between time points.
485 486 487 488
ACKNOWLEDGEMENTS We thank the anonymous reviewers for their comments. This work was
489
supported by the National Key Basic Research Program of China (2013CB430004)
490
and the Basic Research Funding, Free Exploration Projects of Shenzhen Science,
491
Technology and Innovation Commission (No. JCYJ20160530191124115).
17
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492
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Figure. 1 Schematic representation of MeHg and IHg disposition in different compartments of Acanthopagrus schlegeli. k is the model’s intercompartmental rate constant (d-1). Capital letter “M” in the parentheses refers to MeHg and capital letter “I” in the parentheses refers to IHg in the specific compartment.
23
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Figure.2 Concentrations of THg (a, b), MeHg (c, d) and IHg (e, f) in different compartments (blood, intestine, gill, liver and carcass) and whole body of Acanthopagrus schlegeli during exposure (12 d) and depuration (30 d). Data are
-1
Total Hg concentration (ng Hg g F.W)
means ± SD (n = 4).
350 300
400
150
300 100
200
50
100
0
0 6
12
18
24
30
36
42
Blood Carcass Gills
(c)
300
-1
-1
Liver Intestine Whole body
(b)
700
500
200
250
0
6
12
18
24
30
(d)
800
600 500
150
400
36
42
Liver Intestine Whole body
700
200
300
100 200
50
100
0
0
0
IHg concentration (ng Hg g F.W)
800
600
250
0
MeHg concentration (ng Hg g F.W)
Blood Carcass Gills
(a)
90
6
12
18
24
30
36
Blood Carcass Gills
(e)
75
0
42
210
6
12
18
24
30
(f)
42
Liver Intestine Whole body
180 150
60
36
120 45
90
30
60
15
30
0
0 0
6
12
18
24
30
36
42
0
6
Days
12
18
24
Days 24
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30
36
42
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Environmental Science & Technology
Figure. 3 The observed plots and fitted curves of MeHg in blood (a), intestine (b), gill (c), liver (d), and carcass (e) of MeHg-exposed fish (Acanthopagrus schlegeli) during
-1
MeHg concentration (ng g F.W)
exposure (12 d) and depuration (30 d). Data are means ± SD (n = 4).
100
900
(a) Blood
Observed Simulated
80
(d) Liver
750 600
60 450
40 300
20
150
0
0
0
6
12
18
24
30
36
42
0 300
6
12
18
24
30
36
42
18
24
30
36
42
(e) Carcass
250 200 150 100 50
-1
-1 MeHg concentration (ng g F.W) MeHg concentration (ng g F.W)
0
6
400
(b) Intestine 320 240 160 250 80
(c) Gill
200 0 150
0
6
12
18
24
30
36
42
100 50
25
0
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6
12
18
24
Day
30
36
42
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Figure. 4 The observed plots and fitted curves of IHg in blood (a), intestine (b), gill (c), liver (d), and carcass (e) of MeHg-exposed fish (Acanthopagrus schlegeli) during
-1
IHg concentration (ng g F.W)
exposure (12 d) and depuration (30 d). Data are means ± SD (n = 4).
(d) Liver
Observed Simulated
120
16 12
90
8
60
4
30
0 0
0
240
6
12
18
24
30
36
0
42
(b) Intestine
50
6
12
18
24
30
36
42
18
24
30
36
42
(e) Carcass
200
-1
IHg concentration (ng g F.W)
150
(a) Blood
20
40
160 120
30
80 20
40 10
0
-1
IHg concentration (ng g F.W)
0
6
12
18
24
30
36
42
12
18
24
30
36
42
0
6
100
(c) Gill 80 60 40 20 0 0
6
Day
26
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Environmental Science & Technology
Figure. 5 The simulated rates (ng d-1) of uptake (red curve), demethylation (blue curve) and elimination (green curve) of MeHg in Acanthopagrus schlegeli during exposure (12 d) and depuration (30 d).
240
Uptake Demethylation Elimination
-1
MeHg flux rate (ng d )
200 160 120 80 40 0 0
6
12
18
24
30
Day
27
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36
42
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Figure. 6 The simulated MeHg demethylation rates (ng d-1) of the intestine (red curve) and liver (blue curve) in Acanthopagrus schlegeli during exposure (12 d) and
-1
MeHg demethylation rate (ng d )
depuration (30 d).
60 45
Intestine Liver
30 15
3 2 1 0 0
6
12
18
24
30
Day
28
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36
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Environmental Science & Technology
Table 1. Estimated parameters for MeHg distribution and transformation in Acanthopagrus schlegeli exposed to dietary MeHg. Rate Definition
Value ± SDa
k(2,7)
Chyme to gut wall
24 ± 5.2
k(2,1)
Blood to gut wall
15 ± 4.5
k(1,2)
Gut wall to blood
6.5 ± 1.8
k(3,1)
Blood to gill
0.93 ± 0.18
k(1,3)
Gill to blood
0.77 ± 0.14
k(4,1)
Blood to liver
3.5 ± 1.0
k(1,4)
Liver to blood
1.7 ± 0.47
k(1,5)
Carcass to blood
0.031 ± 0.002
k(5,1)
Blood to carcass
4.6 ± 0.42
k(10,7)
Chyme to feces
1.6 ± 1.6
k(20,4)
Demethylation rate in liver
0.011 ± 0.001
k(17,7)
Demethylation rate in chyme
6.5 ± 1.7
constant (d-1)
a
SD = Standard deviation.
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Environmental Science & Technology
Table 2. Estimated parameters for IHg distribution in Acanthopagrus schlegeli exposed to dietary MeHg. Rate constant
Definition
Value ± SD
k(12,17)
Chyme to gut wall
12 ± 5.4
k(12,11)
Blood to gut wall
17 ± 5.2
k(11,12)
Gut wall to blood
2.0 ± 0.57
k(13,11)
Blood to gill
2.7 ± 0.35
k(11,13)
Gill to blood
0.001 ± 0.01
k(14,11)
Blood to liver
0.006 ± 0.01
k(11,14)
Liver to blood
0.23 ± 0.03
k(11,15)
Carcass to blood
0.11 ± 0.03
k(15,11)
Blood to carcass
7.0 ± 1.8
k(10,17)
Chyme to feces
2.0 ± 1.1
k(0,13)
Gill excretion rate
1.1 ± 0.15
(d-1)
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