In Vivo Mercury Demethylation in a Marine Fish - ACS Publications

Article pubs.acs.org/est

In Vivo Mercury Demethylation in a Marine Fish (Acanthopagrus schlegeli) Xun Wang,†,‡ Fengchang Wu,§ and Wen-Xiong Wang*,†,‡ †

Division of Life Science, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong, China ‡ 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 S Supporting Information *

ABSTRACT: Mercury (Hg) in fish has attracted public attention for decades, and methylmercury (MeHg) is the predominant form in fish. However, the in vivo MeHg demethylation and its influence on Hg level in fish have not been well-addressed. The present study investigated the in vivo demethylation process in a marine fish (black seabream, Acanthopagrus schlegeli) under dietary MeHg exposure and depuration and quantified the biotransformation and interorgan transportation of MeHg by developing a physiologically based pharmacokinetic (PBPK) model. After exposure, we observed a 2-fold increase of the whole-body inorganic Hg (IHg), indicating the existence of an in vivo demethylation process. The results strongly suggested that the intestine played a predominant role in MeHg demethylation with a significant rate (6.6 ± 1.7 day−1) during exposure, whereas the hepatic demethylation appeared to be an extremely slow (0.011 ± 0.001 day−1) process and could hardly affect the whole-fish Hg level. Moreover, demethylation in the intestine served as an important pathway for MeHg detoxification. Our study also pointed out that in vivo MeHg demethylation could influence Hg level and speciation in fish although food is the major pathway for Hg accumulation. Enhancing in vivo MeHg biotransformation (especially in the intestine) could be a potential key solution in minimizing Hg contamination in fish. The related factors involved in intestinal demethylation deserve more attention in the future.

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ated)14 and biological processes (microbial activities).15 However, in vivo demethylation in fish has not been welldescribed. Joiris and Holsbeek16 observed that the MeHg ratio in the liver of two sardines decreased with age (from 50% to 20%), suggesting that it might reflect the existence of a slow demethylation process. Based on the quantifications by Hg stable isotopes, however, Wang et al.17 suggested that the decreased MeHg ratio in the liver was likely due to MeHg interorgan transportation from liver to muscle rather than demethylation. Feng et al.18 observed that demethylation occurred in zebrafish (Danio rerio) but could not distinguish the specific organ for this process. It is also debatable where the in vivo MeHg biotransformation occurs. As a detoxification organ, the liver is naturally suspected to be the major site for demethylation, but contradictory results were found in previous studies. Gonzalez et al.19 observed that MeHg represented 66% of mercury in the liver of zebrafish (D. rerio) at day 0 and

INTRODUCTION Mercury (Hg) is a global and highly toxic metal pollutant attracting the world’s attention.1,2 As one of the few metals known to biomagnify along the food chains in aquatic environments, Hg [especially methylmercury (MeHg)] can be easily accumulated and concentrated by fish.3 The elevated levels of MeHg in fish have raised public concern on fish consumption.4,5 It is intriguing that the majority of Hg in fish is presented in methylated form,6,7 although inorganic Hg (IHg) is the predominant form (>95%) in natural water.8 Traditionally, the high levels of MeHg in fish were considered to be derived from trophic transfer9 and attributed to its higher biomagnification potential than IHg.10,11 Indeed, the in vivo MeHg biotransformation (demethylation) can be a potential key process that determines the final biological fate and speciation of Hg in fish.12 Because MeHg could be converted into IHg through demethylation, the occurrence and rate of this reaction would directly affect the relative abundance of IHg versus MeHg in fish. However, this process has not been thoroughly investigated and still remains unclear. In the aquatic environment, demethylation can take place via physical (photodemethylation),13 chemical (selenium-medi© 2017 American Chemical Society

Received: Revised: Accepted: Published: 6441

February 20, 2017 May 12, 2017 May 17, 2017 May 18, 2017 DOI: 10.1021/acs.est.7b00923 Environ. Sci. Technol. 2017, 51, 6441−6451

Article

Environmental Science & Technology

prepared by incubating 100 g of clean commercial fish food with 125 mL of freshly prepared solution (100 μg of MeHg added as MeHgCl). Next, the food pellets were dried at room temperature for 2 days. The measured concentrations of THg and MeHg in the spiked fish diet were 1.07 ± 0.10 and 1.03 ± 0.11 μg g−1 dw, respectively. Dietary Exposure, Depuration, and Sampling. After acclimation in the laboratory, fish were randomly selected and divided into two groups (control and MeHg-exposed group). A total of four aquariums (size of 60 cm × 30 cm × 45 cm) were used for each group, with 20 fish in each aquarium for MeHgexposed group and 4 fish in each aquarium for the control group. The fish in the MeHg-exposed group were fed the MeHg-spiked food pellets, whereas the control group was fed a clean diet during the exposure period. The exposure lasted for 12 days, and feeding was carried out twice a day at a rate of 0.016 g dry weight g−1 wet weight day−1. The diet consumption time lasted for 1 h, and the feeding behavior was monitored to ensure that almost all of the food pellets were eaten (>95%). Next, the uneaten food pellets and feces were siphoned off. After the exposure, the fish were depurated for another 30 days. Fish in both groups were fed clean food pellets at the same rate. During the entire experiment period, fish were kept under the same conditions as those during the acclimation period, and the seawater was cycled at a flow rate of 3 L/min to ensure that the water was clean. The MeHg-exposed fish were sampled at 0, 3, 6, 9, 12, 13, 15, 17, 20, 24, 28, 32, 37, and 42 days, whereas sampling of the control group took place every 6 days. Each aquarium was considered to be one replicate for each treatment, and one fish was randomly collected from each aquarium at each sampling time point. Next, fish were rinsed by deionized water and narcotized in cold ice water. The caudal fin was cut off, and the drained blood was collected by capillary pipet. Fish were then dissected and separated into intestine, gills, liver, and carcass. After the weighing, the fish samples were freeze-dried and stored for further measurements. Chemical and Statistical Analysis. All of the fish samples as well as the fish diet (clean and spiked) were determined for THg and MeHg concentrations. The analysis of THg followed the method of EPA 7474 with a few modifications. Briefly, the homogenized samples (0.05−0.1 g of dw) were digested at 80 °C with 2 mL of aqua regia in a heating block for 12 h. The digested solution was diluted as appropriate. An aliquot of the diluted sample was added into the mixture of hydrochloride, bromate, and bromide to ensure that all forms of Hg were oxidized into Hg(II) ions. Before analysis, samples were reduced by addition of sodium chloride hydroxylamine hydrochloride. THg was then measured by cold-vapor atomic fluorescence spectrometry (CVAFS, QuickTrace 8000, detection limit of liver ≫ gills ≈ intestine > blood, whereas IHg concentrations followed this trend: liver ≫ intestine ≈ gills > carcass > blood. The estimated parameters of MeHg and IHg for different compartments are listed in Tables 1 and 2,

definition

value ± SDa

k(2,7) k(2,1) k(1,2) k(3,1) k(1,3) k(4,1) k(1,4) k(1,5) k(5,1) k(10,7) k(20,4) k(17,7)

chyme to gut wall blood to gut wall gut wall to blood blood to gill gill to blood blood to liver liver to blood carcass to blood blood to carcass chyme to feces demethylation rate in liver demethylation rate in chyme

24 ± 5.2 15 ± 4.5 6.5 ± 1.8 0.93 ± 0.18 0.77 ± 0.14 3.5 ± 1.0 1.7 ± 0.47 0.031 ± 0.002 4.6 ± 0.42 1.6 ± 1.6 0.011 ± 0.001 6.5 ± 1.7

a

definition

value ± SD

k(12,17) k(12,11) k(11,12) k(13,11) k(11,13) k(14,11) k(11,14) k(11,15) k(15,11) k(10,17) k(0,13)

chyme to gut wall blood to gut wall gut wall to blood blood to gill gill to blood blood to liver liver to blood carcass to blood blood to carcass chyme to feces gill excretion rate

12 ± 5.4 17 ± 5.2 2.0 ± 0.57 2.7 ± 0.35 0.001 ± 0.01 0.006 ± 0.01 0.23 ± 0.03 0.11 ± 0.03 7.0 ± 1.8 2.0 ± 1.1 1.1 ± 0.15

MeHg concentrations in whole fish increased greatly from 41 to 214 ng Hg g−1 FW during the exposure period, indicating that MeHg could be easily absorbed and accumulated in fish. More intriguingly, whole-body IHg increased by 2-fold (from 21 to 42 ng Hg g−1 FW) during this period and the newly accumulated IHg (21 ng Hg g−1 FW) accounted for a considerable proportion (>10%) of THg (193 ng Hg g−1 FW) (Figure 2f), demonstrating that IHg was also deposited in fish. Given that MeHg was the only significant source for Hg intake (THg and MeHg in the spiked fish diet were 1.07 ± 0.10 and 1.03 ± 0.11 μg g−1 dw, respectively), our study strongly suggested that demethylation of MeHg occurred in black seabream. These observations can also be supported by modeling results. The MeHg uptake, demethylation, and elimination rates during exposure were estimated to be 180, 50, and 10 ng Hg day−1 (on average), respectively (Figure 5). This suggested that more than 20% of the total ingested MeHg was demethylated into IHg, which subsequently significantly affected Hg composition in fish. Thus, the modeling results pointed out that demethylation was a significant process and played an important role in MeHg disposition. Chumchal et al.36 observed that MeHg comprised the majority of THg in the muscle of spotted gar (Lepisosteus oculatus), whereas IHg was the predominant form in the liver, suggesting that demethylation occurred in this fish species. However, Drevnick et al.39 ascribed the low MeHg and high IHg ratios in the liver of northern pike (Esox lucius) to the IHg uptake from the dietary source but not demethylation. Eagles-Smith et al.40 also observed significant taxonomic differences in demethylation ability in water birds. These observations suggested that the demethylation potential in fish and other vertebrates might be species-specific. Thus, there is a further need to investigate the occurrence of demethylation in other marine fish species. Overall, on the basis of the direct observations and mathematical modeling, we provided direct evidence on the existence of in vivo MeHg demethylation and revealed its importance in the internal handling of MeHg by the marine fish black seabream. During the depuration period, whole-body concentrations of IHg decreased significantly to 19 ng Hg g−1 FW (the same level to the beginning) when depuration ended (Figure 2f). This suggested that the IHg derived from MeHg demethylation could be efficiently eliminated. The elimination rate constant (ke, day−1) for IHg can be calculated as the absolute value of the slope of linear regression of the natural log of the percentage of IHg retained in whole body against depuration time.41 The estimated ke value for IHg was 0.024 ± 0.002 day−1, which was

Table 1. Estimated Parameters for MeHg Distribution and Transformation in A. schlegeli Exposed to Dietary MeHg rate constant (day−1)

rate constant (day−1)

SD: standard deviation.

respectively. The simulated curves for MeHg and IHg in different compartments of MeHg-exposed fish are shown in Figures 3 and 4, respectively. Overall, the model-data plots were well-fitted. The RSDs of most estimated parameters were lower than 0.5, indicating sufficient statistical accuracy. Besides, all of the correlation coefficients between parameters were lower than 0.9, suggesting that all the parameters functioned independently and that the modeling was not overparameterized. The whole-body concentrations were calculated by adding the products of the concentrations of each organ multiplied by its proportion of whole-body weight. As shown in Figure 2b, 6445

DOI: 10.1021/acs.est.7b00923 Environ. Sci. Technol. 2017, 51, 6441−6451

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Figure 3. Observed plots and fitted curves of MeHg in blood (a), intestine (b), gill (c), liver (d), and carcass (e) of MeHg-exposed fish (A. schlegeli) during exposure (12 days) and depuration (30 days). Data are means ± SD (n = 4).

comparable to that measured in the same fish species (0.031 day−1for 15 g of FW fish).11 However, MeHg in whole fish showed no significant change during the depuration period (Figure 2d), indicating that MeHg was difficult to be eliminated and its loss was negligible within the time frame of our study. Traditionally, food-chain transfer is considered to be the predominant pathway for Hg accumulation,3 and the high proportion of MeHg in fish is ascribed to its higher biomagnification potential than IHg.10,11 However, our study showed that there could be a considerable amount of IHg generated from demethylation and accumulated by fish, even if MeHg was the only Hg source. The final high MeHg ratio

(>90%) in whole fish (Figure S2) was caused by the relatively fast elimination of IHg and the extremely slow loss of MeHg. Therefore, our study suggested that the Hg deposited in fish could be derived from varied sources (such as demethylation) rather than from food only. The extremely high MeHg proportion observed in wild fish7,42 could be attributed to a series of complicated physiological-biochemical processes in which the in vivo MeHg biotransformation could make great influence on Hg composition in fish. Given that a significant amount of MeHg was transformed into IHg and could be eliminated within a rather short period, demethylation helped to reduce the accumulation of MeHg and diminish its toxic 6446


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Figure 4. Observed plots and fitted curves of IHg in blood (a), intestine (b), gill (c), liver (d), and carcass (e) of MeHg-exposed fish (A. schlegeli) during exposure (12 days) and depuration (30 days). Data are means ± SD (n = 4).

declined significantly (from 89 to 37 ng Hg g−1 FW) during exposure (Figure 2f), suggesting that the contribution of hepatic demethylation to IHg accumulation in whole fish should be rather limited. On the contrary, IHg in the intestine increased greatly (from 27 to 138 ng Hg g−1 FW) during exposure (Figure 2f) and possessed around 40% of THg at the end of exposure (Figure S2b), suggesting that significant amount of IHg was generated in the gut lumen. Because MeHg was orally taken by fish in this study, the intestinal flora might play an important role in MeHg biotransformation.23 Second, the simulated demethylation rate in the intestine was around 50 ng day−1 (on average) during exposure, whereas that in the liver was only around 1 ng day−1 (Figure 6). This strongly demonstrated that the intestine rather than liver dominated in MeHg demethylation when fish were under MeHg exposure. Previously, Feng et al.18 observed an important contribution

effects on fish. It is considered that fish tend to store the largest amount of MeHg in the muscle, thus protecting other tissues from MeHg toxicity.12 However, this study suggested that demethylation could be another pathway for MeHg detoxification in fish and enabled a better understanding of the detoxification and elimination of MeHg by fish. Demethylation Sites in Fish. The demethylation of MeHg was traditionally suspected to occur mainly in the liver of fish.16,19 However, on the basis of the following two reasons, our study strongly suggested that the intestine was the major site for demethylation when fish were exposed to MeHg. First, the 2-fold increase in whole-body IHg concentration indicated that a large amount of IHg was deposited in fish. If demethylation primarily took place in the liver, there should be a significant amount of IHg produced in the liver, and its IHg level should be greatly elevated. However, liver IHg 6447


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demethylation process, as they might be present in fish digestive tracts. During depuration, IHg concentrations in the intestine declined greatly (Figure 2f). Because fish were fed with clean food within this period, there was no MeHg that could be utilized by intestinal microflora. Thus, intestinal demethylation was stopped, and the liver was the major site for demethylation within this period. It is notable that MeHg concentrations in the liver declined significantly (from 177 to 110 ng Hg g−1 FW) from day 20 to day 42, whereas IHg concentrations increased from 48 to 108 ng Hg g−1 FW (Figure 2d,f). More intriguingly, THg concentrations in the liver were kept stable within this period (Figure 2b). All of these observations suggested that the IHg derived from demethylation was immobilized in the liver and could not be transferred out. Perrot et al.45 also observed that MeHg was demethylated in vivo and that the formed IHg was stored in the liver of mammals. In fish, IHg was found to be co-localized with selenium (Se) in the liver, and a positive correlation between their concentrations was observed,32,46 suggesting that Se might be involved in the demethylation process in the liver. The possible mechanism in hepatic demethylation could be via the formation of HgSe(s),47,48 which is inert and sequestered within hepatic cells.49 However, it should be noted that liver IHg greatly decreased during the first 9 days, suggesting that the original IHg in the liver was “active” and could be transferred out. Because the newly accumulated MeHg tended to be first transferred to the liver, the “active” IHg could be replaced by MeHg and distributed to other parts of fish. To simulate the different behaviors of IHg in the liver, we divided liver IHg into two subcompartments: active pool and storage pool. The former one refers to IHg that can be exchanged with blood, and the latter one represented IHg derived from demethylation and stored in the liver. The simulated IHg mass in the active pool of liver declined in a higher rate than IHg production from demethylation during the initial period (Figure S3). Thus, the modeling successfully described the IHg kinetics in the liver and explained why IHg decreased during the first 9 days and then gradually increased until the end. Given that hepatic demethylation was a rather slow process (the estimated rate constant equaled to 0.011 ± 0.001 day−1) and the formed IHg could not participate in body circulation, its influence on Hg deposition in whole fish was limited in this study. In this study, the methylation process was not considered for the following two reasons. First, the fish were fed with MeHg only; thus, the substrate for the methylation process was not available. Another possibility is that the generated IHg in the intestine lumen might be transformed back into MeHg. However, Lu et al.44 observed that both methylation and demethylation could be carried out by the same anaerobic bacteria; thus, the direction of reaction depends on which species of Hg is mainly provided to the bacteria. Because fish were fed with MeHg-spiked food only, the methylation could hardly occur in the intestine. Second, the methylation of IHg into MeHg in fish is an extremely slow process. Wang et al.17 found that only 0.67−1.60% of the ingested IHg was methylated into MeHg in freshwater fish during the 2 month depuration. Given that the depuration in our study lasted for 1 month, no more than 1% of the generated IHg could be converted back into MeHg. Thus, the influence of methylation on the disposition of IHg and MeHg within the fish body was negligible in our study. Its contribution needs to be further

Figure 5. Simulated rates (ng day−1) of uptake (red curve), demethylation (blue curve), and elimination (green curve) of MeHg in A. schlegeli during exposure (12 days) and depuration (30 days).

Figure 6. Simulated MeHg demethylation rates (ng day−1) of the intestine (red curve) and liver (blue curve) in A. schlegeli during exposure (12 days) and depuration (30 days).

(∼35%) of IHg in the feces resulting from MeHg demethylation in zebrafish (D. rerio) but could not distinguish the demethylation site(s) due to the influence by multiple transport steps. Here, we synthetically considered the processes of demethylation and interorgans transportation and evaluated the contributions of the liver and intestine in demethylation by utilizing PBPK modeling. The results suggested that the intestine not only was the major site for demethylation but also might play an important role in the regulation of Hg level and detoxification of MeHg. Given that significant amount of MeHg was demethylated in the intestine, the decreased MeHg uptake could protect other tissues from its toxicity. The generated IHg from demethylation could be subsequently absorbed, thus greatly affecting Hg composition in fish. However, the related gut microflora and the possible mechanism involved in intestinal demethylation remains unclear. One possible explanation is that the elevated level of MeHg in the gut lumen might induce the expression of genes encoding organomercurial lyase (MerB) and mercuric reductase (MerA),43 thus leading to higher demethylation rates. Some specific strains of anaerobic (e.g., iron-reducing bacteria)44 and aerobic microbes15 could take part in the 6448


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Environmental Science & Technology investigated within a longer time scale or with an elevated IHg level in fish diet. Implication on MeHg Control in Fish. Hg (especially MeHg) in fish, as the most important route for humans exposed to Hg, has raised particular concern to public health for decades.4,50 Decreasing the bioavailability of MeHg has been long-considered to be the major pathway to reduce the MeHg accumulation by fish.12 However, our study found that Hg level and speciation in fish could be greatly affected by in vivo MeHg biotransformation. If the demethylation process could be enhanced within a fish’s body, less MeHg would be accumulated by fish. Compared to the liver, the intestinal demethylation possessed higher potential to be implicated on MeHg control in fish for the following two reasons. First, the intestine (rather than the liver) played a dominant role in MeHg demethylation when fish were exposed to MeHg. Given that the intestine was also the major site for MeHg uptake, intestinal demethylation could decrease the amount of MeHg assimilated by fish, thus helping control of the MeHg accumulation from the source. Besides that, there is no need to concern about the extra IHg uptake derived from intestinal demethylation because IHg could be eliminated within a short period. Second, the influence of hepatic demethylation on the whole-fish Hg level was negligible due to its extremely low rate. For fish, more than 80% of the MeHg body burden is stored in muscle.12 However, the MeHg elimination from muscle is extremely slow, attributed to its tight binding with cysteine-rich proteins.51 Because the MeHg transfer from muscle to liver was rather limited, demethylation in the liver could hardly reduce MeHg accumulation by fish. Overall, our study suggested that enhancing intestinal demethylation could be a potentially useful pathway for MeHg control in fish. The factors that may influence this process (including the specific bacteria strains, temperature, pH, etc.) deserve more investigations in the future. Our study for the first time provided direct evidence on the existence of in vivo MeHg demethylation in a marine fish (A. schlegeli) and quantified the biotransformation and inter-organ transfer processes of MeHg by utilizing PBPK modeling. On the basis of the observations and simulation results, the present study strongly suggested that the intestine played the dominant role in demethylation under MeHg exposure and that intestinal demethylation occurred in a significant rate. Moreover, demethylation in the intestine served as an important pathway for MeHg detoxification. However, hepatic demethylation was an extremely slow process and contributed very little to the whole-body Hg level and speciation. Our study also pointed out that in vivo MeHg demethylation could influence Hg level and speciation in fish, although diet is the major pathway for Hg accumulation. Enhancing in vivo MeHg biotransformation (especially in the intestine) is suggested to be a potential key solution in minimizing Hg contamination in fish. The related factors involved in intestinal demethylation need to be investigated further in the future.

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calibration, equations used for calibrations, and analysis of variance of THg, MeHg and IHg concentrations. (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; phone: (852)-23587346. ORCID

Wen-Xiong Wang: 0000-0001-9033-0158 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the anonymous reviewers for their comments. This work was supported by the National Key Basic Research Program of China (Grant No. 2013CB430004) and the Basic Research Funding, Free Exploration Projects of Shenzhen Science, Technology, and Innovation Commission (Grant No. JCYJ20160530191124115).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.7b00923. Figures showing concentrations of THg and MeHg, MeHg and IHg ratios, and the simulated IHg mass. Tables showing fish weights parameters used for 6449


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