Chemical Forms of Mercury and Selenium in Fish Following Digestion

Fish is the major source of dietary methylmercury species in human ... In particular, it has been suggested that the high selenium levels naturally pr...
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Chemical Forms of Mercury and Selenium in Fish Following Digestion with Simulated Gastric Fluid Graham N. George,*,† Satya P. Singh,† Roger C. Prince,‡ and Ingrid J. Pickering† Department of Geological Sciences, UniVersity of Saskatchewan, 114 Science Place, Saskatoon, Saskatchewan S7N 5E2, Canada, and ExxonMobil Biomedical Sciences Inc., 1545 Route 22 East, Annandale, New Jersey 08801 ReceiVed May 13, 2008

Fish is a major dietary source of potentially neurotoxic methylmercury compounds for humans. It is also a rich source of essential selenium. We have used in situ mercury LIII-edge and selenium K-edge X-ray absorption spectroscopy to chemically characterize the methylmercury and selenium in both fresh fish and fish digested with simulated gastric fluid. For the mercury, we confirm our earlier finding [Harris et al. (2003) Science 301, 1203] that the methylmercury is coordinated by a single thiolate donor, which resembles cysteine, and for the selenium, we find a mixture of organic forms that resemble selenomethionine and an aliphatic selenenyl sulfide such as Cys-S-Se-Cys. We find that local chemical environments of mercury and selenium do not change upon digestion of the fish with simulated gastric fluid. We discuss the toxicological implications for humans consuming fish. Introduction Mercury is widely acknowledged as one of the most toxic heavy elements found in the environment, but its various chemical forms exhibit significantly different toxic properties (1). For example, dialkylmercury derivatives are sufficiently deadly that they have been called “supertoxic” (2), while mercuric selenide has a relatively low toxicity and accumulates as an apparently benign mercury detoxification product in the livers of marine mammals (3, 4). Knowledge of the chemical nature of a potential toxicant is thus essential to predicting its toxic properties. Exposure to toxic levels of methylmercury can result in a variety of pathological outcomes. These can be especially severe when exposure is in utero and can include microcephaly, cerebropalsy, seizures, and mental retardation, among others. Fish is the major source of dietary methylmercury species in human populations. Predatory marine fish such as swordfish and shark contain sufficiently high levels of methylmercury species that consumers are currently advised to eat these fish less frequently than once a month and not at all if pregnant (5). The exact nature of the methylmercury coordination in fish was only recently discovered using in situ X-ray absorption spectroscopy (XAS) (6). XAS is ideally suited to investigations of trace element chemistry in complex biological systems such as whole tissues in that it requires no pretreatment of the sample (7). There are well-established interactions between the toxicology of mercury and selenium in mammals. Ganther and co-workers showed that selenium naturally present in tuna could in part counteract the toxic effects of exogenous methylmercury hydroxide in Japanese quail (8). They also showed that when rats were given both methylmercury hydroxide and sodium selenite, the toxic effects of the mercury were counteracted (8-10). More recent work has emphasized the importance of the protective effects of selenium (11, 12) and has suggested * To whom correspondence should be addressed. E-mail: g.george@ usask.ca. † University of Saskatchewan. ‡ ExxonMobil Biomedical Sciences Inc.

that an important factor in assessing the adverse effects of dietary methylmercury species is the mercury to selenium ratio (11, 12). In particular, it has been suggested that the high selenium levels naturally present in fish might actually serve to counteract the adverse effects of methylmercury (8, 11, 13, 14). The chemical nature of selenium will, however, affect its uptake and availability. We report herein further in situ X-ray absorption spectroscopic studies of the molecular nature of mercury in fish and also examine the molecular nature of the selenium and the molecular fate of both mercury and selenium upon digestion with simulated gastric fluid.

Experimental Procedures Sample Preparation. All reagents were obtained from Sigma Aldrich and were of the best quality available. Fresh fish samples (swordfish, Xiphias gladius) were obtained at a local fish market from fish that was otherwise destined for human consumption. Slices of fish were cut with a scalpel and loaded into acrylic XAS sample cuvettes and frozen in liquid nitrogen prior to data acquisition. Simulated gastric fluid was prepared according to the U.S. Pharmacopoeia (15) using porcine pepsin. A 100 mg amount of swordfish skeletal muscle was finely chopped using a scalpel and was incubated with 0.5 mL of simulated gastric fluid at 37 °C for 1 h. The pH of the solution was monitored at 10 min intervals during the digestion and kept between 1.0 and 2.0 by adding small quantities of hydrochloric acid. At the end of the digestion, the tissues had broken down and the sample had been converted to a milky-gray liquid. This material was loaded into acrylic XAS sample cuvettes using a hypodermic syringe and frozen in liquid nitrogen prior to data acquisition. Samples of bis(methylmercuric)selenide and methylmercury-selenocysteinate were prepared according to published methods (16, 17). XAS. XAS measurements were conducted at the Stanford Synchrotron Radiation Laboratory (SSRL) with the SPEAR storage ring containing between 90 and 100 mA at 3.0 GeV. Mercury LIIIedge and selenium K-edge data were collected on the structural molecular biology XAS beamline 9-3 operating with a wiggler field of 2 T, employing a Si(220) double-crystal monochromator. Beamline 9-3 was equipped with a rhodium-coated vertical collimating mirror upstream of the monochromator and a downstream bent-cylindrical focusing mirror (also rhodium-coated). Harmonic rejection was accomplished by setting the cutoff angle of the mirrors

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Chemical Forms of Mercury and Selenium in Fish to 15 keV. To minimize radiation damage, samples were maintained at a temperature of 10 K or below in an Oxford instruments flow cryostat. X-ray absorption spectra were measured as the Se K R1,2 or Hg L R1,2 fluorescence excitation spectra using a 30 element germanium array detector (18) with analogue electronics (Canberra Corp.) employing an amplifier shaping time of 0.125 µs. To avoid problems with nonlinearity of the detector, X-ray filters (made of elemental As for Se and Ga2O3 for Hg) were used to preferentially absorb scattered radiation, with silver Soller slits (EXAFS Co., Pinoche Nevada) optimally positioned between the sample and the detector. Incident and transmitted XAS intensities were measured using nitrogen-filled ionization chambers. The mercury spectra were energy-calibrated with reference to LIII-edge spectrum of Hg-Sn amalgam foil measured simultaneously with the data, the lowest energy inflection of which was assumed to be 12285.0 eV. The inflection of Hg-Sn amalgam was determined to be identical with that of a microparticulate aqueous suspension of pure metallic mercury precipitated from aqueous HgCl2 by reduction with a slight excess of sodium borohydride. The selenium spectra were similarly energy calibrated with reference to the spectrum of hexagonal elemental selenium at 12658.0 eV. For the Hg data of intact fish, 11 scans were averaged, while 21 scans were accumulated for the more dilute digested fish sample (each of 18 min duration). Four scans were averaged for the selenium data of intact fish (each of 21 min duration), while six scans were averaged for the digested fish. XAS data were processed using standard techniques and employing the EXAFSPAK program suite (19), and spectroscopic reproducibility was found to be excellent (Figure S1 of the Supporting Information). Near-edge spectra were fitted to linear combinations of standard spectra using the EXAFSPAK program DATFIT, which minimizes the sum of squares difference between the experimental and the simulated data. In general, components were rejected when the fraction of the component was less than the 99% confidence limit (given by 3× the estimated standard deviation from the diagonal elements of the covariance matrix). Tests of the curve-fitting protocol were conducted on simulated spectra synthesized by adding the spectra of standard compounds and incorporating computer-generated random noise at the various signal-to-noise levels. These tests indicated that the detection of minor components in mixtures depended upon both the similarity of the spectra of the components and the signal-to-noise levels of the data. These tests indicated a worst-case scenario for minor component detection by our curve-fitting procedure at or below the 10% level at the levels of noise that are present in our data.

Results and Discussion Chemical Forms of Hg and Se in Fish. X-ray absorption spectra arise from excitation of a core electron (e.g., a 1s electron for a K-edge or a 2p3/2 electron for an LIII-edge). They can be arbitrarily divided into two overlapping regionssthe near-edge spectrum, which is the structured region within approximately 50 eV of the absorption edge, and the extended X-ray absorption fine structure (EXAFS), which is an oscillatory modulation of the absorption on the high-energy side of the absorption edge and which can be interpreted in terms of a local radial structure. Near-edge spectra are comprised of transitions from the core level (1s for a K-edge) to unoccupied molecular orbitals of the system. Intense transitions are dipole-allowed ∆l ) (1, and thus for K and LIII edges are to levels with a lot of p and d orbital character, respectively. Near-edge spectra are therefore sensitive to electronic structure and give a fingerprint of the chemical species of the metal or metalloid concerned. The advantage of the near-edge region of the spectrum is that it can be quickly collected with good signal-to-noise. In contrast, EXAFS is more difficult to collect with adequate signal-to-noise and is not always practical on dilute samples. A unique benefit of XAS is that it requires no pretreatment or extraction and thus provides a tool that can probe chemical species in situ. Figure

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Figure 1. Comparison of Hg LIII and Se K near-edge spectra of selected standard species. CH3HgSe(Cys), (CH3Hg)2Se, and HgSe were run as solids by monitoring transmittance, while other samples were prepared as dilute (ca. 1 mM) aqueous solutions buffered at physiological pH (7-7.5) in the presence of 30% v/v glycerol and were measured using X-ray fluorescence.

1 shows the Hg LIII and Se K near-edge spectra of selected standard compounds. The differences between individual Hg LIII spectra are more subtle than for the Se K spectra. In the case of Se K-edges, the valence orbitals have a lot of p character, giving rise to intense dipole-allowed structure in the spectra and rich chemical variability, whereas for Hg LIII edges, there are only subtle involvements of d orbitals and correspondingly more subtle variability between the spectra. The consequence of this is that significantly better signal-to-noises (when measured relative to the edge jump) are required for Hg than for Se, but the spectra can nevertheless give the desired information. Figure 2 shows the Hg LIII and Se K X-ray absorption nearedge spectra of swordfish skeletal muscle. For mercury (Figure 2A), the spectrum is essentially identical to that reported previously (6) and, as before, indicates that the mercury is predominantly by a single methyl group and a single aliphatic thiol such as cysteine (6). As discussed above, the spectroscopic variability of mercury is less than that observed for selenium, and the chemical effects in the spectra are more subtle (Figure 1). As we have discussed, the signal-to-noise requirements for the Hg LIII near-edge spectra are therefore correspondingly more stringent. Figure 3 shows a superimposition of the Hg LIII nearedge spectra of selected standard compounds with that of fish, and as previously reported (6), the spectrum of methylmercury cysteine is the only one that clearly matches. Curve-fitting analysis using the set of spectra shown in Figure 1A indicates just a single component, with no significant contributions from any other species (a table of fit errors is given as Supporting Information in Table S1, two-component fits statistically rejected the second component). For selenium (Figure 2B), the nearedge spectrum shows a superficial resemblance to that of selenomethionine, but fitting of a linear combination of model spectra indicates the presence of other components at significant levels, in particular a selenenyl sulfide such as Cys-S-Se-Cys, plus smaller amounts of an inorganic component resembling selenite. We note that in general, near-edge spectra cannot distinguish between species with similar atomic neighborhoods. Thus, selenium compounds with similar aliphatic ligands (e.g., Se-methylselenocysteine and selenomethionine) have essentially identical spectra (20). As a consequence of this, we can conclude

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Figure 2. (A) Mercury LIII near-edge spectrum of intact swordfish skeletal muscle (points) as compared with the spectrum of methylmercury L-cysteine. The spectra are identical within the noise. Single component fits to the nine spectra shown in Figure 1 are tabulated in the Supporting Information (Table S1). (B) Selenium K near-edge spectrum of swordfish skeletal muscle (points) as compared with a leastsquares fit from a linear combination of model compound spectra. The three significant components are plotted below for comparison selenomethionine (s), 52 ( 3%; Cys-Se-S-Cys (- - -), 38 ( 3%; and selenite (· · ·), 10 ( 1% (errors are estimated standard deviations obtained from the diagonal elements of the covariance matrix).

Figure 3. Comparison of the mercury LIII X-ray absorption nearedge spectra of swordfish with spectra of selected standard compounds. The points show the spectrum of the fish, while the solid lines show the spectra of the standard species (all conditions are as for Figures 1 and 2).

that the chemical nature of selenium coordination is (for example) selenomethionine or a selenomethionine-like species, but no specific molecular identity can be established. No signs of the HgSe-like phases found in marine mammals and birds

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were observed (3, 4, 21, 22). Elemental concentrations can be estimated directly from X-ray absorption spectra by comparing the edge jumps from un-normalized background-subtracted spectra with edge jumps measured from spectra of solutions containing the element of interest (either Se or Hg) at known concentrations. When this is done, we obtain approximate concentrations of 5.0 and 5.1 µM for Hg and Se, respectively. These values are essentially slightly lower (for Hg) than those reported earlier (6) and very similar to those reported by Kaneko and Ralston for pacific Swordfish (13), indicating that they fall within the expected range. The fact that Hg and Se are approximately stoichiometric also doubly reinforces our finding that no significant levels of HgSe compounds are present in the fish, as the presence of these would be reflected in both the Se K and the Hg LIII spectra. The observation that methylmercury is bound to a single thiol is not surprising in view of mercury’s well-known affinity for thiol groups, and such coordination was widely assumed, although unproven prior to our earlier work (6). Methylmercury is known to exchange rapidly with thiols (23), presumably because of the thermodynamic stability of three-coordinate species, which have been recently observed by mass spectrometry (24)

CH3HgSR + R’SH T [CH3Hg(SR)SR’]- + H+ T CH3HgSR’ + RSH According to some reports (25), the high chloride concentrations and low pH of the stomach might convert the methylmercury species present in fish to methylmercury chloride:

CH3HgS(Cys) + H+ + Cl- f CH3HgCl + (Cys)SH CH3HgCl is a hydrophobic molecule that should readily cross membranes to be absorbed into the body through the gastrointestinal tract (25). This postulated chemistry has implications for the absorption of methylmercury in the stomach. Figure 4 compares the Se K and Hg LIII X-ray absorption near-edge spectra of fresh and digested swordfish. The spectra of undigested and digested fish are obviously similar, as illustrated by the difference spectra shown in Figure 4. These were generated by subtracting the pairs of Se and Hg spectra, which essentially consist only of noise. This striking result indicates that digestion using simulated gastric fluid does not cause significant changes in the local chemical environments of either mercury or selenium. Thus, in contrast to previously reported work (25), the high chloride and low pH of gastric fluid do not result in formation of CH3HgCl, and either the reported equilibria (25) are inaccurate or the other components of this complex system such as digested protein or hydrophobic compounds (fats) significantly interfere. Insofar as selenium is concerned, the lack of changes in the aliphatic selenide component (modeled as selenomethionine) is not particularly surprising from a chemical standpoint, but our data also indicate that the selenenyl sulfide component remains molecularly intact and that no significant chemistry involving both Hg and Se occurs following digestion of fish. Toxicological Implications for Human Consumption of Fish. Considerable publicity has been given recently to the possible health hazards associated with eating fish. The current recommendations are based in part upon data from different populations that ingest large quantities of mercury from seafood (26, 27), along with the results of a number of animal studies. The two largest studies are of primarily fish-eating people in

Chemical Forms of Mercury and Selenium in Fish

Figure 4. (A) Mercury LIII X-ray absorption near-edge spectra of intact swordfish skeletal muscle (a) and the product of its digestion with simulated gastric fluid (b). The traces have been vertically offset for clarity. The essential identity of the two spectra is illustrated by the difference spectrum (b - a). (B) Selenium K near-edge spectra from the samples used for panel A.

the Seychelles (26) and of people who eat a mixed whale meat and fish diet in the Faroes (27). In both of these independent ongoing studies, correlations were sought between mercury exposure in utero and subsequent neurobehavioral deficits in children. The conclusions of the two groups are somewhat at odds. The Faroes study (27) has consistently indicated a correlation of neurobehavioral deficits with maternal mercury levels when the subject was in utero, while the Seychelles study (26) showed no effects. In setting the limits for human consumption, the Faroes study was preferentially selected, to avoid possible adverse consequences of setting limits too high (28). An obvious shortcoming of this is that the Faroes diet contains significant whale meat and blubber. These contain other toxic species that are not found in fish such as cadmium in meat (29) and polychlorinated dibenzodioxins in blubber (30), which are not found in the diets of most other humans. Thus, it may be that while mercury correlates with measurable neurobehavioral deficits, the actual culprit may be another toxin that is present in whale, but not in fish, that in turn correlates with the mercury burden. Alternatively, the chemical form of mercury in whale meat may differ from that of fish in terms of its bioavailability. Clearly, more studies of whale are needed before definitive conclusions can be formed, and this will be the subject of future work. We have suggested previously that ingestion of a mercury dose through consumption of fish, which contains methylmercury cysteine, may have quite different toxicological implications than ingestion of the same mercury dose in the form of other methylmercury compounds (6). Norseth and Clarkson have reported that following administration of methylmercury chloride to rats, methylmercury cysteine is excreted in bile (31), and they have suggested that methylmercury cysteine cycles through enterohepatic circulation, finally exiting in feces via sloughing of intestinal lumen cells. This proposal seems very reasonable in light of the fact that methylmercury cysteine is known to be transported by the LAT1 and LAT2 amino acid transporter (32) and likely excreted (as glutathione conjugates) by the multidrug

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resistance proteins MPR1 or MPR2 (33). Berntssen and coworkers (34) have reported that rats fed high-mercury fish show higher fecal excretion and lower tissue accumulation of mercury than rats consuming fish to which methylmercury chloride was added artificially. Our finding that methylmercury cysteine present in fish is not transformed into the chloride by simulated gastric fluid is consistent with the findings of Berntssen and co-workers (34). Possible Protective Effects of Selenium in Fish. Watanabe et al. (35) and subsequently Ralston and co-workers (12) have suggested that the mechanism for the neurotoxic effects of methylmercury compounds has its basis in the development of local selenium deficiencies in the brain. Ralston et al. (12) demonstrated that rats exposed to methylmercury had very low brain selenium levels. In contrast, animals exposed to both selenium and mercury retained more normal levels of brain selenium and were protected from the toxic effects of mercury (12). Ralston and co-workers postulate that following methymercury transported to the central nervous system the mercury is demethylated to form inorganic mercury that reacts with brain selenium to effectively create brain selenium deficiency and that this is the mechanism of the neurotoxic effects observed for methylmercury compounds (12). While this mechanism is at present conjectural, it is very clear that, as discussed above, selenium can protect against the toxic effects of methylmercury in mammals (8, 11-14). Conversely, Reed et al. (36) investigated the effects of selenium (given as sodium selenite in food) on adult rats exposed to methylmercury in utero (given as CH3HgCl in drinking water) and found no evidence for any interactions. Using the Faroes cohort, Grandjean and co-workers (37) have measured both Hg and Se levels in cord whole blood. They examined the data both for effects of selenium upon neurodevelopmental deficits and for correlations between mercury and selenium levels. They failed to detect either effect and concluded that there was no evidence for a protective effect of selenium (37), at least when the selenium was measured in cord blood, and assuming that the neurodevelopmental deficits are indeed directly related to mercury. However, selenomethionine has recently been demonstrated to reduce visual deficits induced by methylmercury in zebrafish (14), which is a common model organism for vertebrate development. Thus, it is not at present clear whether or not there are measurable adverse effects due to mercury in fish (from uncontaminated waters), nor is it clear whether selenium can protect against mercury’s effects, if they occur. Our finding that the selenium in fish is predominantly in organic forms means that the selenium from fish will likely be readily absorbed and metabolized. Whether or not the selenium can protect against methylmercury may well depend upon the molecular form of both the metal and the metalloid. In summary, we have compared fish before and after digestion in simulated gastric fluid and have found that both mercury and selenium species are unchanged by the treatment. This indicates that the more toxic methylmercury chloride will not be produced in the stomach. Likewise, selenium in the fish is predominantly in organic forms, which will be readily absorbed and metabolized. Acknowledgment. This work was supported by a grant from the Canadian Institutes of Health Research. Research at the University of Saskatchewan was supported by Canada Research Chair awards (to G.N.G. and I.J.P.), the University of Saskatchewan, the Province of Saskatchewan, the National Institutes of Health, and the Natural Sciences and Engineering Research Council (Canada). Portions of this work were also carried out at the Stanford Synchrotron Radiation Laboratory,

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which is funded by the U.S. Department of Energy, Office of Basic Energy Sciences, and Office of Biological and Environmental Sciences, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program. We thank members of the George/Pickering research group for contributions to data collection. Supporting Information Available: Spectroscopic reproducibility at the Hg LIII edge (Figure S1) and Table S1, which shows fit errors for Hg LIII near-edge spectra fish. This material is available free of charge via the Internet at http://pubs.acs.org.

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