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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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Environmental Science & Technology

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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: [email protected] phone: (852) 23587346

1

ACS Paragon Plus Environment


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ABSTRACT Mercury (Hg) in fish has attracted public attention for decades, and

20

methylmercury (MeHg) is the predominant form in fish. However, the in vivo MeHg

21

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,

24

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

33

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)

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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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3



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INTRODUCTION

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Mercury (Hg) is a global and highly toxic metal pollutant attracting the world’s

43

attention.1,2 As one of the few metals known to biomagnify along the food chains in

44

aquatic environments, Hg [especially methylmercury (MeHg)] can be easily

45

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

50

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

52

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

65

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

68

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

70

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

101

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

150

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

177

be determined. A compartment is a theoretical construct that may include several

178

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

180

experimental data, and the structure of the model is then altered until the fitting on the

181

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

183

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

188

Hg.27

189 190 191

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

193

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

201

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

205

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

210

taken up (Flux(12,17)). The residual MeHg and IHg in the chyme were finally

211

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

217

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

220

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

223

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

227

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

230

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

234

pool at time t. wliver refers to the fresh weight of the liver. It should be noted that the

235

“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

237

blood.

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Data fitting. We used the SAAM II modeling software version 2.3.1 (SAAM 9



239

Institute, University of Washington, Seattle, WA, USA) to construct the modeling and

240

calculate the kinetic parameters. SAAM II has been successfully used to simulate the

241

distribution and elimination of Hg as well as other trace metals within fish body.27,37

242

The modeling structure was constructed based on two guiding principles: it should

243

contain the fewest compartments to adequately describe the data; it should reflect the

244

realistic physiology or metabolic process. Thus the final modeling structure was

245

reached by a process of trial. During the fitting process, the parameters were given

246

initial values comparable to published data on MeHg and IHg distribution in fish, and

247

were allowed to vary until the best fitting was reached. The software would finally

248

give the mean and the standard deviation (SD) of the parameters. To provide the best

249

fit, the software could iteratively minimize an objective function based on the Akaike

250

information criterion (AIC) and the Schwarz–Bayesian information criterion (BIC).

251

These two criteria are a function of the goodness of fit, the number of adjustable

252

parameters, and the total number of data points. When comparing two potential

253

modeling structures, lower values of AIC and BIC indicated the modeling better

254

described the data with the least number of parameters.37 The quality of parameter

255

estimates were evaluated based on SDs of the parameters, the parameter correlation

256

matrixes and the appearance of the data-model plots. If the RSD (the ratio of the

257

standard deviation to the mean value) was lower than 0.5, the parameter would be

258

considered to be different from zero with 95% confidence. If the correlation

259

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

261

overparametrized.38

262 263 264

RESULTS AND DISCUSSION

In vivo demethylation of MeHg. In this study, the concentrations of THg and

265

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

268

(MeHg) and inorganic mercury (IHg) in the five compartments (blood, intestine, gills,

269

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

274

stable until the end of experiment. For carcass, THg declined slightly but MeHg

275

showed no obvious change. Moreover, THg in the liver decreased dramatically until

276

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

278

concentrations in the blood, intestine, gills and carcass increased significantly during

279

exposure, then decreased continuously during depuration and reached to the same

280

level as the initial values at the end of experiment. However, IHg in the liver showed

281

an opposite trend, which declined sharply (F = 21.2) and reached to the bottom at the

282

end of exposure, then increased greatly (F = 29.6) during the last 30 days. At the end

283

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

286

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.

289

The RSDs of most estimated parameters were lower than 0.5, indicating sufficient

290

statistical accuracy. Besides, all of the correlation coefficients between parameters

291

were lower than 0.9, suggesting that all the parameters functioned independently and

292

the modeling was not overparameterized.

293

The whole-body concentrations were calculated by adding up the products of the

294

concentrations of each organ multiplied by its proportion of whole-body weight. As

295

shown in Fig. 2b, MeHg concentrations in whole fish increased greatly from 41 to 214

296

ng Hg g-1 F.W (fresh weight) during the exposure period, indicating that MeHg could

297

be easily absorbed and accumulated in fish. More intriguingly, whole-body IHg

298

increased by 2-fold (from 21 to 42 ng Hg g-1 F.W) during this period and the newly

299

accumulated IHg (21 ng Hg g-1 F.W) accounted for a considerable proportion (> 10%)

300

of THg (193 ng Hg g-1 F.W) (Fig. 2f), demonstrating that IHg was also deposited in

301

fish. Given that MeHg was the only significant source for Hg intake (THg and MeHg

302

in the spiked fish diet were 1.07±0.10 µg g-1 dw and 1.03±0.11 µg g-1 dw,

303

respectively), our study strongly suggested that demethylation of MeHg occurred in

304

black seabream. These observations can also be supported by modeling results. The 11



305

MeHg uptake, demethylation and elimination rates during exposure were estimated to

306

be 180, 50 and 10 ng Hg d-1 (on average), respectively (Fig. 5). This suggested that

307

more than 20% of the total ingested MeHg was demethylated into IHg, which

308

subsequently significantly affected Hg composition in fish. Thus, the modeling results

309

pointed out that demethylation was a significant process and played an important role

310

in MeHg disposition. Chumchal et al.36 observed that MeHg comprised the majority

311

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

313

species. However, Drevnick et al.39 ascribed the low MeHg and high IHg ratios in the

314

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

317

demethylation potential in fish and other vertebrates might be species specific. Thus,

318

there is a further need to investigate the occurrence of demethylation in other marine

319

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

321

revealed its importance in the internal handling of MeHg by the marine fish black

322

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

330

same fish species (0.031 d-1for 15 g F.W fish).11 However, MeHg in whole fish

331

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

339

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

341

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



371

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


Page 14 of 30

Page 15 of 30


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



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


Page 16 of 30

Page 17 of 30


470

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



492

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22


Page 22 of 30

Page 23 of 30


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



Page 24 of 30

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


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


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


30

36

42

Page 25 of 30


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

ACS Paragon Plus Environment 0

6

12

18

24

Day

30

36

42

12


Page 26 of 30

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


150

(a) Blood

20

40

160 120

30

80 20

40 10

0

-1


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


12

Page 27 of 30


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


36

42


Page 28 of 30

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


36

42

Page 29 of 30


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.

29



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)

30


Page 30 of 30

In Vivo Mercury Demethylation in a Marine Fish ... - ACS Publications

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