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Title: The Molecular Basis for Mercury Toxicity Long-Term Objectives: To understand the mechanism of mercury toxicity at a molecular level, how this is influenced by other chemical species such as selenium, and to design more effective chelation therapy drugs. Background Mercury poisoning is a health concern. Mercury’s toxicity is well-known and worries many communities in Canada and worldwide.1,2 Human exposure to mercury comes from many sources. Methylmercury compounds are known to be highly neurotoxic, and are present in significant quantities in predatory marine fish (swordfish, shark etc.).2 In a recent US EPA/FDA advisory (the first joint advisory ever) pregnant women, women who might become pregnant, and nursing mothers are advised to completely avoid high-mercury fish such as swordfish, king mackerel, or tilefish.3 Other sources of exposure include silver-mercury amalgam fillings in teeth, and the organo-mercury compound thimerosal (ethylmercurithiosalicylate). Thimerosal has been added as a fungicide and bactericide to many vaccines since the 1930’s, and recently has been suggested to be a possible cause of autism and Asperger’s syndrome.4,5 Environmental mercury exposure can also be a major problem; two examples are gold mining operations in Brazil, and effluent from the pulp and paper industry in Ontario. Large-scale catastrophic outbreaks of mercury poisoning have occurred in Japan and Iraq.6 These tragic incidents amply demonstrate the insidious and debilitating nature of mercury poisoning – its creeping neurotoxicity, particularly in children.1,6,7,8 A plethora of other complaints have also been reported from mercury poisoning – central nervous system defects, arrhythmias and cardiomyopathies, and kidney damage. Inhalation of mercury can result in necrotizing bronchitis and pneumonitis which in turn can result in respiratory failure.1,6,7,8 Mercury can also act as either an immunostimulant or an immunosuppressant, depending on the nature of the exposure, leading to a number of pathologic consequences.1,6,7,8 At some level, mercury exposure and potential toxicity is a concern for all Canadians, whether from fish in their diet, or from other sources of exposure. Molecular form matters. As with all heavy metals, the nature and the extent of the toxicity of mercury varies tremendously depending on its molecular form. Some compounds, for example dimethylmercury, are toxic at such low levels that they are considered “supertoxic”,9 while others, such as mercuric sulfide, are relatively benign, and sufficiently inert to be used in jewelry. The toxicology of mercury also appears to be intimately tied to the biochemistry of selenium, which can effectively cancel out or magnify (and modify) its toxic properties.10,11 Conventional analytical techniques will often modify the chemical form of mercury from the form found in situ, and thus direct information about the molecular mechanisms of mercury toxicity is lost. A technique that can reveal molecular form in situ is thus expected to be of vital importance in understanding molecular mechanisms. There are many potential chemical forms of mercury in situ. Mercury is well-known for its affinity for thiols, and because of this many suggestions for mechanisms involve coordination of an essential thiol, usually as a simple two-coordinate complex. In fact, mercury has a highly variable coordination chemistry. For example, while coordination of Hg2+ by thiolates is favored thermodynamically, stable complexes can be achieved with either two [Hg(SR)2], three [Hg(SR)3]-, or four [Hg(SR)4]2- coordination.12 Likewise, methylmercury* can potentially coordinate one, two or three thiolate ligands * There is a slightly confusing nomenclature that is common in the literature. According to standard chemical nomenclature “methylmercury” is what is commonly called dimethylmercury (CH3HgCH3). Here we adhere to the common usage in which methylmercury contains only a single methyl, with one or more other ligands to the mercury (e.g. CH3Hg–R) . It is also common practice to denote aqueous

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(Figure 1). Examination of the Cambridge Structural Database13 indicates that coordination numbers between two and eight are common, and nitrogenous or oxygen donors are also possible. Mercury also forms strong bonds with selenium, and mercury-metal bonds can be particularly stable. X-ray absorption spectroscopy provides an in situ probe for mercury chemical identity. X-ray absorption spectroscopy (XAS) can provide information on the chemical environment of metals and metalloids in situ, with no pre-treatment of the sample. Until recently XAS has lacked the sensitivity to measure physiologically relevant levels, with millimolar concentrations typically being required for adequate signal to noise.14 Recent developments in detector technology and modern high-flux X-ray beamlines have allowed our group to lower this threshold to the sub-micromolar domain (Figure 2).15,† This provides new opportunities for the application of XAS to in situ investigations of mercury molecular toxicology, and this is the foundation of this proposal. Important groundwork for our proposed studies is provided by our experience with XAS of intact plant tissues,16,17,18,19 and our preliminary studies on mammalian systems.10,20 The application of XAS to the study of metabolites in intact tissues is relatively new. To our knowledge, there are no other groups anywhere in the world attempting to address toxicological questions using X-ray absorption spectroscopy. Our proposed research will thus represent an important milestone for biomedical applications of synchrotron radiation, quite apart from the biomedical benefits likely to grow from our work. Available chelation therapy drugs are not well optimized. One goal of our work is to provide the chemical basis for more effective chelation therapy treatments of mercury poisoning in humans. Currently, two different drugs are used for mercury chelation therapy – dimercapto propanesulfonic acid (DMPS), and dimercapto succinic acid (DMSA). Both of these vicinal dithiol drugs have their origins in antidotes for arsenic war agents such as Lewisite (chlorovinylarsinedichloride). While both drugs are effective at some level in mercury chelation therapy,21,22 we have recently shown that they are poorly optimized for their clinical function of binding mercuric ions as they cannot actually bind as chelators.23,‡ Previous work has demonstrated that they are of little use in treating poisoning from organo-mercury compounds24 – a striking illustration was the tragic case of Karen Wetterhahn, a chemist at Dartmouth College USA, who was accidentally exposed to a small quantity of dimethylmercury. Despite intensive chelation therapy, Dr. Wetterhahn died nearly ten months after her exposure.9

Custom chelator design using molecular modelling. Computer-aided drug design is increasingly common in modern medical science. Density functional theory (DFT) is among the most powerful tools available to the computational chemist, and the recent availability of these efficient, rigorous and powerful modern codes has revolutionized the field of quantum chemistry (Walter Kohn shared the 1998 Nobel Prize in Chemistry with John Pople for the development of DFT). Unlike older codes, DFT can be used to compute ab initio the three dimensional structures of molecules involving any atom (older codes cannot reliably handle heavy atoms, such as mercury). Knowledge of the chemical forms of

solutions of methylmercury compounds, for example methylmercury chloride, as containing CH3Hg+ but we note that this cation will probably never actually exist in solution, and in the case of chloride the Cl will remain strongly bound to Hg in solution. † Levels of mercury are often quoted in parts per billion (ppb), 1 µM corresponds to 200 ppb. Our most dilute measurements to date have been around 0.2 µM or 40 ppb, and this should improve with anticipated increases in X-ray photon flux and refinements in technique. ‡ A chelate is a complex in which a ligand (the chelator) forms two or more bonds to a single metal or metalloid ion.

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mercury in tissues is an essential prerequisite for chelation therapy drug design, and we plan to use the information obtained from XAS to this end. Our approach will be to employ modern DFT codes to design new chelator molecules which will structurally “fit” only mercury: “custom chelators”. Overall Goal 1. Elucidate the molecular basis of mercury toxicity. This will be approached by using X-ray absorption spectroscopy to study tissues and fluids of mammals injected with mercury, the mercury in dietary fish before and after digestion, and tissues from deceased humans with a high-fish diet. Overall Goal 2. Design new chelation therapy molecules – “custom chelators” – to bind mercury strongly and specifically. This will be approached using modern density functional theory codes to design the custom molecules, in conjunction with XAS to test the results. Overall Strategy. Herein we propose to employ X-ray Absorption Spectroscopy in a broad-based approach, including studies of animal tissues, high mercury foods such as fish, and human tissue, to obtain a molecular-level understanding of mercury toxicology, and to explore potential remediation strategies (custom chelators). This is an ambitious and aggressive proposal, in which we plan to deploy powerful new experimental techniques and computational methods against the problem of mercury poisoning. Experimental Methods The technique. X-ray absorption spectroscopy (XAS) (Figure 3) is used to determine local physical and electronic structures of specific metals or metalloids. As a technique it is very well developed, but it has almost never been applied to problems in molecular toxicology. XAS can be divided into two regions; the near-edge spectrum and extended X-ray absorption fine structure (EXAFS) (Figure 4). Near-edge spectra, comprising the region within about 50 eV of the absorption edge, are dominated by dipole-allowed transitions to vacant molecular orbitals, and thus contain information on electronic structure. The near-edge spectrum of an unknown can essentially be used as a “fingerprint” of chemical form by comparison to a library of model compound spectra, and a quantitative understanding of the transitions observed in near-edge spectra is not necessary to identify chemical type (Figure 5). EXAFS is the extended structure which continues above the absorption edge, generally requiring more signal averaging to obtain adequate signal to noise, but yielding detailed local structural information. XAS requires no pre-treatment of the sample, can be done on any physical form, and is thus readily applied to intact tissue samples. An estimation of the concentration of mercury can be made from the size of the edge-jump. The bulk of the work described in this proposal will employ near-edge spectra “fingerprints” because the dilute nature of many of the samples will preclude collection of EXAFS with adequate signal to noise. Mercury LIII near-edge spectra typically lack the intense absorptions seen for many other elements (e.g. selenium K-edges) which means that better signal to noise is typically required to use the fingerprinting technique at the mercury LIII edge. Fortunately, this is counterbalanced by the fact that Hg LIII XAS spectra can be collected at better signal to noise than e.g. selenium because the Hg Lα1 fluorescence line (the primary LIII fluorescence) is separated from the absorption edge by a rather larger energy (e.g. 2.3 keV vs. 1.4 keV for the Se K-edge). The consequence of this is that there is essentially no superimposed background from the low-energy tail of the inelastically (and to a lesser extent elastically) scattered radiation at the Hg Lα1 energy, which significantly adds to the background at the Se Kα energy. The improvements in signal to noise (statistical noise is proportional to the square root of total counts) and low background allow us excellent sensitivity at the Hg LIII edge. Model compounds. In order to establish the feasibility of using the Hg LIII near-edge spectrum to “fingerprint” the chemical identity of Hg in tissue samples, we have already examined the spectra of a

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number of relevant low molecular weight species of known structure – “model compounds” – and a selection of these is shown in figure 5. As expected, the spectra are sensitive to the chemical environment of the metal, but (also as expected) somewhat less so than for some other elements, such as Se and As. In some cases we may need to use the EXAFS portion of the spectrum to more unambiguously identify the mercury coordination. We plan to record the spectra of additional mercury model compounds to further develop the “fingerprint” library of Hg LIII near-edge XAS spectra. The extreme toxicity of some mercury compounds mandates great care in the handling and disposal of these compounds, and we will work with the University of Saskatchewan and Synchrotron Safety personnel to ensure both personnel and environmental safety. X-ray absorption spectroscopy imaging. XAS imaging is a new technique in which a small beam is obtained using microfocus optics,25,26 and the sample is raster scanned using a high precision sample stage while monitoring the X-ray fluorescence of a particular element (e.g. the mercury Lα1) so that an image of the mercury (or whatever element is being studied) levels in a sample can be built up.17,25,26

This is a new form of microscopy (Figure 7), and the co-applicant and applicant developed the experimental apparatus that is available at the Stanford Synchrotron Radiation Laboratory (SSRL).

Experimental Setup. These experiments will be done at the Canadian Light Source (CLS) XAFS beamline which is due to be commissioned in late 2004. Until the CLS XAFS beamline is available we will use SSRL structural molecular biology XAS beamlines 7-3 and 9-3. A highly-rated beamtime proposal describing this work is active (SSRL Proposal No. 2818 “The Molecular Basis for Mercury Toxicity”; G. N. George, Spokesperson). The dilute nature of our samples requires the use of a 30-element Ge array detector system, two of which are available at SSRL. A similar detector will be purchased for use at the CLS using the combined CFI CRC chair allocations (approved) of G. N. George and I. J. Pickering, with matching funds from the Province of Saskatchewan, and contributions from both the University of Saskatchewan and the vendor. All measurements will be done in Oxford Instruments Liquid Helium Cryostats. SSRL currently has six of these, and the applicant currently has full discretionary use of one additional cryostat (on permanent loan from ExxonMobil Research and Engineering Company) currently located at SSRL but which could be brought to the CLS. A CFI application for funds for a dedicated Oxford Cryostat system for the CLS XAFS beamline is pending under the ongoing new opportunities fund (project No. 6630, H. Nichol, principal proponent). Facilities for XAS Imaging are presently available at SSRL, and similar capabilities are planned for the CLS XAFS beamline (we note that the significantly increased flux that will be available at the CLS should allow the collection of better data). An Oxford Cryosystems Helix helium cryostat is available at SSRL for cooling XAS Imaging samples. Our proposed work involves tissues obtained from animal studies but no animal work will be conducted in our laboratory at the University of Saskatchewan, CLS or SSRL. All animal work will be performed at the Institute for Ecological Chemistry by our collaborators, and will be subject to that institute’s regulations. Samples will be loaded in XAS sample holders, will be shipped frozen in liquid nitrogen to the University of Saskatchewan, the CLS or SSRL (as appropriate), and then returned for appropriate disposal. The Hg LIII near-edge spectra will be examined to identify the mercury species present and the total mercury concentration (from the edge-jump), and if concentration permits we will also record the EXAFS (as we have discussed, better signal to noise is required for EXAFS). Analysis of mixtures. Many of the samples we plan to measure are expected to contain predominantly a single species, but some may be more complicated. The analysis of mixtures is more challenging than a single species, but we have developed methods using least-squares fitting of linear combinations of

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models16,27 that are now standard in the field. More recently we have applied more sophisticated numerical methods such as principal component analysis28 to define the number of components present in a set of data. As we have noted above, mercury LIII near-edge spectra are broader than those from other elements, and resolution of a complex mixture of species may not be possible. Nevertheless, we are as well or better equipped than any other investigators to attack such a problem and we have the technical expertise to know whether or not it can be solved. Molecular modelling. Density functional theory molecular modeling will use the program Dmol3 with the Materials Studio graphical interface available from Accelrys Corp.29,30 We plan to follow the same basic methods as in our previous work,23 in which the Becke exchange31 and Perdew correlation32 functionals were used to calculate both the potential during the SCF, and the energy. We will use double numerical basis sets including polarization functions for all atoms, calculations will be spin-unrestricted, and all-electron relativistic core potentials will be employed. Optimized geometries will typically use energy tolerances of 2.0 x 10-5 Hartree. Research Plan Overall Goal 1. Elucidate the molecular basis of mercury toxicity. Objective 1.1: Identify the molecular entities formed in mammalian tissues upon administration of mercury compounds. Methylmercury compounds are much more toxic to mammals (and in particular neurotoxic) than mercuric salts, but in cell cultures methylmercury compounds are about as toxic as mercuric salts.33 It is thought this difference might arise from the increased ability of methylmercury to cross membranes, but there are other perplexing aspects of the toxicology. Methylmercury compounds exhibit a significant latency between the administration and the onset of toxic symptoms,34 which can be as long as 150 days.9 The cause of this latency remains one of the major unanswered questions in this field. Previous work has suggested that demethylation occurs with time,35 and it has been supposed that the gradual accumulation of Hg2+ species is responsible for the latency period. Methylmercury compounds have been shown to trigger apoptosis in cultured dorsal root ganglion neurons,36 and interactions with Ca2+ channels and oxidative stress may be involved,37 and links with glutamate metabolism have been suggested.38 Weiss, Clarkson and Simon have recently reviewed this area and suggest three scenarios.34 Methylmercury in the brain may be acting as a trigger; causing catastrophic cell death once its concentration in the brain reaches a certain threshold level. Alternatively, the mercury could trigger synthesis of a protective molecule, which finally becomes exhausted at the end of the latent period. Weiss et al. also suggested a different scenario – that the latency may be due to the presence of populations of neuronal cells with differing susceptibilities. The more susceptible cells would die first, being functionally replaced by the less susceptible cells. With time, the latter also succumb because of increased functional stress or metabolic load.34 Alternative scenarios can also be envisaged; if supporting cells such as glia are more susceptible to mercury than the neurons that depend on them, then these neurons will die only after glial cell death, causing a latent period in the development of symptoms. Hwang et al. have proposed a hypothesis for the mechanism of methylmercury toxicity, based on their observations that the upbiquitin-proteosome pathway is involved in methylmercury resistance in yeast and human cells.39,40 They propose that methylmercury binds an unknown protein (which they call X) that then becomes cytotoxic.40 Nothing is known about protein X, except that it contains a signal for ubiquitination by the enzyme Cdc34. The mode of mercury binding is unknown – it might be to a cysteine thiolate, but it might equally be to a metal ion. Thus, the mechanisms behind methylmercury neurotoxicity remain unknown, and knowledge of the molecular identities of mercury in central nervous system tissues will provide an important part of the puzzle. In collaboration with Dr. Jürgen Gailer of Boehringer Ingelheim Austria and his collaborators at the GSF National Research

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Center for Environment and Health, Neuherberg, Germany, we propose to examine the XAS of tissue samples and excretory products of rabbits treated with different mercury compounds, and determine the chemical forms of mercury in vivo. Primary target organs for mercury (and in particular methylmercury compounds) are brain and kidney, and these, together with other tissues, will be examined as a function of the administered form of mercury, of time and of dose. In certain cases (see objective 1.4, below) we will administer mercury compounds orally. This should facilitate an understanding of the molecular species that form in the toxic response. Three mercury compounds will initially be studied, mercuric chloride, methylmercury cysteine, and thimerosal. Our preliminary experiments have shown that some mercury compounds are modified soon after exposure. Figure 6 compares the EXAFS Fourier transforms of a thimerosal standard with rabbit kidney 30 minutes after intravenous injection of thimerosal. The EXAFS indicate that the thimerosal is substantially and rapidly chemically modified in vivo. The EXAFS are consistent with an additional longer oxygen ligand to Hg forming the stalk of a T-shaped complex. Significant short-term accumulation in the brain was also observed (up to 3.2 µM until observations were stopped at 60 min.) although biochemical transformation was not observed in the brain. Our XAS studies of mercury biochemical transformation are expected to provide insights into the basic molecular mechanisms behind mercury toxicity. Objective 1.2: Determine the molecular basis of the antagonism and the synergism between mercury and selenium in mammals. The importance of antagonistic and synergistic relationships between toxic heavy elements and the essential (but also toxic) element selenium is increasingly recognized. Mercury compounds show a curious time-dependent relationship with selenium. If CH3Hg-R or Hg2+ complexes are administered at approximately the same time as an equal dose of selenite, the toxic effects of the mercury are relieved. We have previously studied the antagonism between mercuric chloride and selenite in rabbits by Se K and Hg LIII XAS and shown that mercuric selenide clusters rapidly form in the blood10 HgSe formation may be a major Hg detoxification mechanism in whales, which are exposed to large quantities of dietary Hg, and crystalline mercuric selenide has been identified in whale liver.41 However, returning to land mammals, if the selenite is pre-administered by ca. 2 hours then the toxic effects are hugely magnified.11 In this, synergistic, case the selenium is presumably converted into a different species (possibly methylated) which then interacts with the mercuric salts in an unknown manner to form a much more potent toxin. Tantalizing preliminary animal studies indicate that when selenite and methylmercury hydroxide are co-administered to rabbits, adverse neurological side effects immediately occur (i.e. convulsions), despite an overall relief of toxicity. We propose to study the mechanism of both the synergistic and antagonistic interaction between mercury compounds and selenium compounds. Key tissue samples (e.g. blood, kidney, liver, spleen, brain) will be studied by both Hg LIII-edge and Se K-edge XAS, with different chemical forms of mercury (mercuric chloride, methylmercury species, etc.), and selenium (selenite, methylselenol, dimethylselenide etc.), as a function of time and of dose. We will be guided by preliminary animal experiments, as only some of the possible permutations can be studied by XAS in the finite amount of beamtime available. Objective 1.3: Determine the localization of mercury within mammalian organs. The bulk XAS studies described above should provide information on chemical form averaged over a piece of tissue (generally about 3mm x 1mm x 2mm). XAS imaging is a new technique in which a small beam is obtained using microfocus optics,25,26 and the sample is raster scanned while monitoring the X-ray fluorescence of a particular element (e.g. mercury) so that the beam interrogates a particular region of the sample . In suitable cases, carefully selected X-ray energies can provide sensitivity to chemical form – allowing collection of a chemically specific image.17 Figure 8 shows preliminary Hg Lα1 XAS image data of 4 day old zebrafish larvae exposed to 100µM methylmercury cysteine for 1 hour (a non-lethal

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exposure). Clear localization of mercury in various tissues of the zebrafish larvae can be observed. The chemical sensitivity observed in Hg LIII near-edge spectra is generally insufficient to allow the resolution of discrete chemical forms by XAS imaging. However, we note that this is not true of selenium for which excellent chemically resolved images can be obtained.17 We propose to apply XAS imaging to the most interesting tissues identified under objective 1.2. We will investigate the spatial localization within sections (ca. 100 µm thick) or, where appropriate, whole organs. Mice will be used for experiments involving intact organs, since the larger size of rabbit organs would cause X-ray beam attenuation problems, and in initial experiments we plan to use imaging at intermediate resolution (ca. 25-15 µm) to allow us to image an entire brain or kidney in a reasonable time-frame (for example 2 hours). This should allow us to determine the spatial localization within the tissue. The feasibility of these experiments will critically depend on the concentration of mercury in the tissues, and we note that for some imaging experiments (e.g. of rabbit brain) we currently lack the sensitivity to detect the mercury, unless there are regions of high concentration. In others tissues, for example kidney and liver, the mercury is expected to be more readily detectible.§ This elemental map of mercury can be further probed by selecting a pixel and recording a near-edge spectrum, which will give information on the chemical identity at that location. For experiments involving selenium, chemically resolved images using more than one X-ray energy may be possible. We note that the direction of these experiments will critically depend upon our findings with bulk XAS (described above). During the active period of this proposal we anticipate that sensitivity limits will continue to improve. The degree to which this will occur is difficult to predict, but it is expected from improvements in detector technology (the new detector that will be deployed at the CLS will have slightly better performance than existing models). Perhaps more significant will be improvements in beamline optical stability, and increases in photon flux. The XAFS beamline at the CLS will be powered by a 64 pole superconducting wiggler magnet operating near 2 T, and this will provide a particularly intense beam (conservatively estimated as ~1.2x1013 photons/sec/mm2 at the Hg LIII-edge). It may thus be possible to perform high resolution studies at 1 µm or better at the CLS within the funding period of this proposal.** Such studies should provide information on the subcellular localization of mercury in large cell types such as neurons. Chronic toxicity is ultimately a subcellular event, and therefore it is important to know in which organelles mercury accumulates and how its chemical form changes during cellular metabolism. These studies will provide a foundation that will be vitally important in developing a molecular understanding of mercury toxicology, and of possible treatment strategies. Objective 1.4: Identify the molecular forms of mercury in dietary fish. In humans, the primary dietary source of mercury compounds is via ingestion of methylmercury species accumulated in fish.1,2 Methylmercury ingestion from fish has been linked to neurological damage (Minamata disease)42 and increased risk of myocardial infarction43, although this is controversial.44 And this has necessarily been balanced against the well-known health benefits of fish consumption (e.g. high ω3 fatty acid content).

§ A 30 min. exposure of rabbits to thimerosal (10 mg/kg) accumulated 580 µM in the kidney from a blood level of 47 µM, 175 µM in the liver, but only 2.5 µM in the brain. ** The beam size enabled by the Kirkpatrick-Baez microfocus optics on the CLS XAFS beamline is predicted to be 2x4 µm, with a flux of 7 x 1010 photons/sec. within that aperture. Significantly smaller X-ray beams could be achieved by the additional use of micro-focussing optics, such as tapered capillaries, or zone plates.

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Two large-sample studies of communities with high levels of consumption of mercury from fish have been reported, both studies examined the links between prenatal mercury exposure and intellectual function in childhood. One of these, based in the Faroe islands in the North Sea, concluded that adverse effects were observed.44 The other, based in the Seychelle islands in the Indian Ocean, concluded the reverse, that no adverse effects could be observed.45 Thus, the epidemiological data is apparently in conflict. Differences in diet (including micronutrients) may be important,46 for example the Faroe islanders consume whale meat (known to contain high levels of mercury), and the Seychelle islanders do not. The latter probably also consume more tropical fruit, which has been reported to reduce uptake of methylmercury from fish.47 The debate continues.48,49 There is no debate, however, about the severe pathological consequences of consumption of fish that have been contaminated with mercury. Mercury contamination of fish is a problem in several areas of Canada. In many cases Aboriginal communities have been the most affected.50

As we have discussed above, knowing the chemical nature of a potential toxicant is essential to understanding its toxic properties, and we have recently investigated the chemical form of methylmercury in marine fish using X-ray absorption spectroscopy (Figure 9).15 We studied swordfish (Xiphias gladius), orange roughy (Hoplostethus atlanticus) and sand sole (Psettichthys melanastictus), although good quality data were not collected for the latter because of limited beamtime. Our findings indicate that the methylmercury is bound to a single aliphatic thiol – which is probably cysteine, and which in turn may be bound as part of a protein or peptide. This is chemically reasonable in view of the stability of the Hg-S bond. In separate experiments on zebrafish larvae we found that methylmercury cysteine was much less toxic than methylmercury chloride.15 This work received widespread press coverage, and unfortunately some members of the media erroneously interpreted our study (and in a few cases misquoted us) as supporting consumption of high Hg fish. The implications of our work for human consumption of fish are, at present, unknown. We propose to extend our earlier studies to other species of predatory fish (e.g. tilefish, Lopholatilus chamaeleonticeps, and shark of various species), including freshwater species (e.g. Northern Pike, Esox lucius). Our earlier work included one low mercury species (sand sole), which contained approximately 0.4 µM mercury (80 ppb). Such concentrations are near to our current lower limit, but with signal averaging adequate data should result, and we propose to study other low mercury fish (including both marine and freshwater species) to ascertain whether the chemical form of Hg is the same. We will also examine Hg contaminated fish to ascertain the chemical form of methylmercury that accumulates when mercury levels are very high. Finally, we will investigate the mercury in lobster and crayfish muscle and mid-gut gland or tomalley (the latter is known to absorb other substances of environmental concern such as poly chlorinated biphenyls). With the sole exception of the Hg contaminated fish, all fish samples will be obtained from local fish markets, and will be material that would otherwise be intended for human consumption.

The acidic high Cl- conditions in the human stomach may convert methylmercury cysteine

species into the chloride, but whether this actually occurs is at present unknown. We will investigate this possibility by spectroscopically examining the stomach and intestinal contents of rats that have been fed high mercury fish (e.g. swordfish). The products of test-tube simulations of mammalian digestion of fish using commercially available gastro-intestinal enzymes (e.g. pepsin for stomach) will also be examined. Once the molecular form in digested fish is determined, then this species will be the subject of tissue studies as described under objective 1.1.

Considerable publicity has been given to possible health hazards associated with eating fish and

our studies will add a much needed chemical perspective to the current state of the art. Objective 1.5: Identify the molecular forms of mercury in human tissues. Because the ultimate goal of our research is a molecular understanding and treatment of mercury poisoning in humans, it is perhaps

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of the greatest interest to know the chemical form of mercury in human tissues. We therefore plan such a study in collaboration with Dr. Gary Myers, MD and co-workers at the University of Rochester Medical Center, in New York, USA. Myers and co-workers have well established collaborations with the pathology department of the Ministry of Health in the Republic of Seychelles. At present samples from the Faroe islands are not available to us, but we will explore this possibility through our collaborators at the University of Rochester. These experiments will thus follow protocols and procedures that are already in place. As noted above, the population of the Seychelles has been extensively studied and found to have elevated mercury in the diet.45 We propose to study the chemical forms of mercury in fresh human brain tissue (i.e. without chemical preservatives). The samples will be frozen in liquid nitrogen shortly after they become available through autopsy in the Seychelles. They will then be transported by our collaborators to Rochester, and stored there in preparation for our next allocation of beamtime, at which time they will be brought to either SSRL or CLS. Samples will be loaded and sealed into sample cuvettes by our Rochester collaborators (this will be done either in the Seychelles or at Rochester) and will be returned for appropriate disposal following XAS data acquisition. Previous studies by Myers, Clarkson and co-workers have shown that brain samples contain mercury in the range 25-300 ppb (0.125-1.5µM), and samples of both high and low mercury content will be examined. Mercury content in hair has been shown to be an excellent indicator of exposure, and we will examine samples of the hair of the same and of other individuals. We note that these samples will be experimentally trivial by comparison due to their much higher mercury content; typically 3-12 ppm (15-60µM). We will also routinely examine the Se K-edge of all samples, which might give indications as to whether this element is involved in mercury binding in the brain.†† As our studies progress it may become of greater interest to examine tissues other than brain and hair, and if so we will perform these experiments. Correlation of these results with the animal work (described above) will provide essential information about whether the animal studies are providing an accurate model of the biochemical transformations of mercury in a real human population. Overall Goal 2. Design new mercury chelation therapy molecules – “custom chelators”. Objective 2.1: Determine the chemical factors that are important in mercury coordination. The most used chelation therapy drugs are meso-DMSA (2,3 dimercaptosuccininc acid) and DMPS (2,3 dimercaptopropane 1-sulfonic acid), which are commercially sold as Chemet® and Dimaval®, respectively. We have recently studied the aqueous solution chemistry of the interaction between Hg2+ and DMSA and DMPS using Hg LIII XAS and density functional calculations.23 We have shown that neither DMSA nor DMPS forms a true chelate‡ with mercury, and we have argued that they are chemically sub-optimal by a significant margin for their task of binding mercury in the body.23 With DMSA at stoichiometries of 1:1 DMSA:Hg and above, a two-coordinate linear S-Hg-S species is formed, and the most likely product contains two Hg atoms and two DMSA molecules (Figure 10).23 With DMPS a similar two-coordinate linear species is also formed at 1:1, but unlike DMSA, higher stoichiometries yield a four coordinate species with characteristic Hg-S bond lengths of about 2.51 Å. This indicates (i) that the solution chemistry chelation products of DMSA and DMPS are significantly different, and (ii) with both chelators, complexes in which the Hg coordination is fully satisfied by DMSA and DMPS only form when more than one molecule of the drug is involved.23 Our work went on to use density functional theory to compute the chemical factors that are most important in the binding of sulfur donors to mercuric ions for the linear two-coordinate species CH3S-Hg-SCH3 in an attempt to derive general rules about mercury coordination. The important factors are the Hg-S bond length (2.35

†† In an unrelated series of experiments, we have already collected selenium K-edge XAS data on untreated hamster brain samples and thus anticipate that the levels of selenium in human brain will be detectable using our methodology.

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Å), the C-S-Hg bond angle (103º), and the C-S-(Hg)-S-C torsion angle (90º). The S-Hg-S bond-angle can vary between 170º and 190º with little energetic effect and is thus less important (Figure 11). A molecule containing a framework that could hold the thiolate groups in the ideal proximity and orientation for mercuric ions would bind mercury immensely more strongly than would other molecules.23 It would also be highly specific for mercury, as other metals prefer a different coordination. We note that this is very similar to the mechanisms employed by biological systems to confer strong binding and specificity. Thus, the prokaryotic metalloregulatory protein of the mercury-resistance operon, known as MerR, will bind Hg2+ with incredible potency, exhibiting K0.5~10-7M in the presence of 10mM β-mercaptoethanol,51 suggesting that Hg2+ binding to MerR is about 100,000 times more effective than to β-mercaptoethanol. MerR appears to bind mercury in a trigonal planar coordination to three cysteine ligands.52,53 At present, effective framework structures have yet to be designed, although it is clear that this is an achievable goal (Objective 2.3). We propose to use density functional calculations to systematically investigate the structural factors that are important in mercury coordination in three-coordinate and four-coordinate mercury thiolate complexes, and two, three and four coordinate mercury selenolate complexes. As with the two-coordinate thiolate we will start with the simple methanethiolate complexes, and then extend the work to larger molecules. Again, as with the two-coordinate thiolate species,23 we will use the Cambridge Crystal Structure Database13 to search for structures in order to verify the validity of our calculations. Calculations will use both simple mercuric ions and methylmercury, together with any mercury species found to be important in our tissue experiments (Objectives 1.1-1.5). Objective 2.2: Investigate the interaction of mercury compounds and potential ligand species in aqueous solution. The computational studies described under objective 2.1 will proceed in parallel with XAS studies of the aqueous solution chemistry of a range of simple and more complex molecules. Typical questions that we will address include: How do DMPS and DMSA interact with different methylmercury species in solution? How do mercuric ions and different methylmercury species interact with other species such as organic selenols and dimethylselenide? Of course, the chemical questions to be asked will depend critically on the results of our calculations (objective 2.1), and of our tissue experiments (Objectives 1.1-1.5), and where possible the results of the solution chemistry experiments will be checked computationally, and vice versa. We note that these experiments will not use just XAS spectroscopy, and, as appropriate, we will also bring more conventional laboratory analytic methods to bear (e.g. mass spectrometry, vibrational spectroscopy, chromatography etc.) in order to obtain as complete as possible understanding of the solution chemistry. Objective 2.3: Design a more effective prospective drug for mercury chelation therapy. Once the chemical forms in situ and the critical factors governing mercury coordination are well understood we may be in a position to design a custom chelator. As described above this would be a molecule containing a framework that could hold the groups to ligate the mercury in the ideal proximity and orientation for binding. This is the ultimate goal of our work. Such a “custom chelator” molecule would not only bind mercury incredibly tightly, but also with a very high degree of specificity. For example, linear two-coordinate complexes are also stable for Cu+ but with a characteristic bond-length to sulfur of 2.15 Å,13 and thus Cu+ would fit a custom chelator designed to bind Hg2+ (bond length to S of 2.35 Å) significantly less well. In our work to date we have examined the digonal thiolate complexes, and while we have yet to design effective framework structures, it is clear that this is an achievable goal. Binding energies are difficult to compute, but an approximate value can be obtained from the difference in computed energies‡‡ between the component ions or molecular fragments and the bound species, for

‡‡ In density functional theory total energies are computed with reference to the energy when all electrons and nuclei are separated to infinity.

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example Hg2+ and CH3S-, and CH3SHgSCH3 (which in this case comes out to 627 kcal/mol). Binding energies computed in this way will be used to predict the effectiveness of candidate custom chelators. One digonal thiolate candidate molecule is shown in Figure 12, in which thiol groups are separated by twice the linear Hg-S bond length. We note that functional groups such as carboxylic or sulfonic acid moieties may need to be incorporated into the molecule to confer the correct balance of water and lipid solubility (the latter to allow the molecule to cross cell membranes). Finally (and we note that this last objective may not be fully realized within the period of this proposal) the most promising candidate molecules will be synthesized by a custom synthesis company (Target Molecules Ltd.), and their solution chemistry with appropriate Hg compounds tested using XAS. These compounds will then be tested by our collaborators in animal studies for both toxic side-effects and effectiveness as chelation agents, and the chemical identity of any Hg remaining in tissues will be determined by XAS. Significance and Future Directions This is an ambitious and aggressive proposal, in which we plan to deploy powerful new synchrotron in situ experimental techniques and modern computational methods against the problem of mercury poisoning. Our work will provide a fundamental molecular understanding of the nature of mercury poisoning, and new, highly specific and effective drugs for removing mercury from humans – “custom chelators”. Our methods could in principal be applied to any metal ion, and our long-term plans include such directions. Obvious candidates for such work include other toxic heavy metals, such as cadmium and lead, or even actinides, whose biological effects and transformations are as yet poorly understood. Aside from clinical use, a variety of additional applications can be considered for a successful custom chelator. For example, custom chelator molecules could be immobilized by attachment to a solid phase and used to remove trace levels of metal ions from contaminated waters (including wastewater treatment plant effluents). Or, in the case of actinides, even as part of an integrated treatment and clean-up plan for the aftermath of an attack with a so-called “dirty bomb”. Dissemination The results of this work will be disseminated by publication in the peer-reviewed scientific literature, and by presentations at international conferences. Additionally the Applicant and Co-Applicant are in the early stages of planning a workshop on biological applications of synchrotron radiation which will include possible applications in the field of molecular toxicology. Any XAS analysis software developed under this proposal will be made available to the academic community free of charge as part of the Applicant’s EXAFSPAK software analysis suite (http://ssrl.slac.stanford.edu/exafspak.html).

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References 1. Clarkson, T. W., Magos, L., and Myers, G. J. “The Toxicology of Mercury – Current Exposures and

Clinical Manifestations” (2003) N. Engl. J. Med. 349, 1731-1737. 2. Goyer, R. A., Aposhian, H. V., Arab, L., Bellinger, D. C., Burbacher, T. M. et al. (2000)

“Toxicological Effects of Methylmercury” National Research Council, National Academy Press, Washington DC.

3. http://www.fda.gov/oc/opacom/mehgadvisory1208.html 4. Bernard, S., Enayati, A., Redwood, L., Roger, H., Binstock, T. (2001) “Autism: a novel form of

mercury poisoning” Med. Hypoth., 56, 462-471. 5. Bernard, S., Enayati, A., Roger, H., Binstock, T. and Redwood, L. (2002) “The role of mercury in

the pathogenesis of autism” Mol. Psych., 7, S42-S43. 6. Clarkson, T. W. (1998) “Human Toxicology of Mercury” J. Tr. Elem. Exp. Med. 11, 303-317. 7. Clarkson, T. W. (2002) “The Three Modern Faces of Mercury” Env. Health Persp. 110 (suppl. 1),

11-23. 8. Tchounwou, P. B., Ayensu, W. K., Ninashvili, N., and Sutton, D. (2003) “Environmental Exposure

to Mercury and Its Toxicopathologic Implications for Public Health” Env. Toxicol. 18, 149-175. 9. Nierenberg, D. W., Nordgren, R. E., Chang, M. B., Siegler, R. W., Blayney, M. B., Hochberg, F.,

Toribara, T. Y., Crenichiari, E., and Clarkson, T. (1998) “Delayed Cerebellar Disease and Death after Accidental Exposure to Dimethylmercury” New England J. Med. 338, 1672-1676.

10. Gailer, J. George, G. N., Pickering, I. J., Madden, S, Prince, R. C., Yu, E. Y., Denton, M. B., Younis H. S. and Aposhian, H. V. (2000) “The Structural Basis of the Antagonism between Inorganic Mercury and Selenium in Mammals.” Chem. Res. Toxicol. 13, 1135-1142.

11. Magos, L. “Overview on the protection given by selenium against mercurials.” In Advances in Mercury Toxicology (Suzuki, K., Imura, N., and Clarkson, T.W. Eds) (1991) pp 283-298, Plenum Press, New York.

12. Govindaswamy, N., Moy, J., Millar, M. and Koch, S. A. (1992) “A Distorted [Hg(SR)4]2- Complex with Akanethiolate Ligands: The Fictile Coordination Sphere of Monomeric [Hg(SR)x] Complexes” Inorg. Chem. 31, 5343-5344.

13. Allen, F. H., Kennard, O. and Watson, D. G. (1994) “Crystallographic databases: Search and retrieval of information from the Cambridge Structural Database” Struct. Correl. 1, 71-110.

14. Scott, R. A. (1984) “X-ray absorption spectroscopy” in Structural and Resonance Techniques in Biological Research. Rousseau, D. L. Ed. Academic Press, Orlando, Florida, USA, pp. 295-362.

15. Harris, H. H., Pickering, I. J., and George, G. N. (2003) “The Chemical Form of Mercury in Fish” Science, 301, 1203.

16. Pickering, I. J., George, G. N., Yu, E. Y., Brune, D. C., Tuschak, C., Overmann, J., Beatty, J. T., and Prince, R. C. (2001) “Analysis of Sulfur Biochemistry of Sulfur Bacteria Using X-ray Absorption Spectroscopy” Biochemistry 40, 8138-8145.

17. Pickering, I. J., Prince, R. C., Salt, D. E., and George, G. N. (2000) “Quantitative chemically-specific imaging of selenium transformation in plants.” Proc. Natl. Acac. Sci. U.S.A. 97, 10717-10722.

18. Pickering, I. J., Prince, R. C., George, M. J., Smith, R. D. George G. N. and Salt, D. E. (2000) “Reduction and Coordination of Arsenic in Indian Mustard” Plant Physiol. 122, 1171-1177.

19. Yu, E. Y., Pickering, I. J., George, G. N., and Prince, R. C. (2001) “In-situ observation of the generation of isothiocyanates from sinigrin in horseradish and wasabi.” Biochim. Biophys. Acta 1527, 156-160.

20. Gailer, J., George, G. N., Pickering, I. J., Prince, R. C., Ringwald, S. C., Pemberton, J. C., Glass, R. S., Younis, H. S., DeYoung, D. W. and Aposian, H. V. (2000) “A Metabolic Link Between As(III) and Se(IV) : The Seleno-bis(S-glutathionyl)Arsinium Ion.” J. Am. Chem. Soc., 122, 4637-4639.

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21. Böse-O'Reilly, S., Drasch, S., Beinhoff, C., Maydla, S., Voskob, M. R., Roidera G. and Dzajaa, D. (2003) “The Mt. Diwata study on the Philippines 2000 – treatment of mercury intoxicated inhabitants of a gold mining area with DMPS (2,3-Dimercapto-1-propane-sulfonic acid, Dimaval®)” Sci. Tot. Env. 307, 71-82.

22. Garza-Ocanas, L., Torres-Alanis, O. and Pineyro-Lopez, A. (1997) “Urinary mercury in twelve cases of cutaneous mercurous chloride (calomel) exposure: effect of sodium 2,3-dimercaptopropane-1-sulfonate (DMPS) therapy” J. Toxicol., Clin. Toxicol. 35, 653-655.

23. George, G. N., Prince, R. C., Gailer, J., Buttigieg, G. A., Denton, B., Harris, H. H. and Pickering, I. J. “Mercury Binding to the Chelation Therapy Agents DMSA and DMPS, and the Rational Design of Custom Chelators for Mercury.” Chem. Res. Toxicol. (submitted)

24. Pfab, R., Mueckter, H., Roider, G. and Zilker, T. (1996) “Clinical course of severe poisoning with thiomersal” J. Toxicol., Clin. Toxicol. 34, 453-460.

25. Pickering, I. J., Hirsch, G., Prince, R. C., Sneeden, E. Y., Salt, D. E. and George, G. N. (2003) “Imaging of selenium in plants using tapered metal monocapillary optics” J. Synchrotron Rad. 10, 289-290.

26. Bertsch, P. M. and Hunter, D. B. (2001) “Applications of synchrotron-based X-ray microprobes” Chem. Rev. 101, 1809-1842.

27. George, G.N., Gorbaty, M.L., Kelemen, S.R. and Sansone, M. (1991) “Direct determination and quantification of sulfur forms in coals from the Argonne premium sample program.” Energy and Fuels 5, 93-97.

28. Principal Component Analysis – near-edge spectra Wasserman? 29. Delley, B. (1990) “An all-electron numerical method for solving the local density functional for

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7764. 31. Becke, A. D. (1988) “A multicenter numerical integration scheme for polyatomic molecules” J.

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correlation energy” Phys. Rev. B 45, 13244-13249. 33. Christensen, M. M., Ellermann-Eriksen, S., Rungby, J., and Mogensen, S. C. (1993) “Comparison of

the interaction of methylmercury and mercuric chloride with murine macrophages.” Arch. Toxicol., 67, 205-211.

34. Weiss, B., Clarkson, T. W., and Simon, W. (2002) “Silent Latency Periods in Methylmercury Poisoning and in Neurodegenerative Disease” Env. Health Persp. 110 (suppl. 5), 851-854.

35. Vahter, M., Mottet, N. K., Fridberg, L., Lind, B., Shen, D. D., and Burbacher, T. (1994) “Speciation of Mercury in the Primate Blood and Brain Following Long-Term Exposure to Methyl Mercury” Toxicol. Appl. Pharmacol. 124, 221-229.

36. Wilke, R. A., Kolbert, C. P., Rahimi, R. A., Windebank, A. J. (2003) NeuroToxicol. 24, 369-378. 37. Gasso, S., Cristofol, R. M., Selema, G., Rosa, R. and Rodriguez-Farre, E. (2001) “Antioxidant

Compounds and Ca2+ Pathway Blockers Differentially Protect Against Methylmercury and Mercuric Chloride Neurotoxicity.” J. Neurosci. Res. 66, 135-145.

38. Qu, H., Syversen, T., Aschnew, M., and Sonnewald, U. (2003) “Effect of methylmercury on glutamate metabolism in cerebellar astrocytes in culture” Neurochem. Intl. 43, 411-416.

39. Furuchi, T., Hwang, G-W., and Naganuma, A. (2002) “Overexpression of the Ubiquitin-Conjugating Enzyme Cdc34 Confers Resistance to Methylmercury in Saccharomyces cerevisiae” Mol. Pharmacol. 61, 738-741.

40. Hwang, GI-W., Furuchi, T., and Naganuma, A. (2002) “A ubiquitin-proteasome system is responsible for the protection of yeast and human cells against methylmercury” FASEB J. 16, 709-711.

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41. Martoja, R., and Berry, J. P. (1980) “Identification of Tiemannite as a probable product of demethylation of mercury by selenium in cetations. A complement to the scheme of the biological cycle of mercury” Vie Milieu 30, 7–10.

42. Kudo, A., Fulikawa, Y., Miyahara, S., Zheng, J., Takigami, H., Sugahara, M. and Muramatsu, T. (1998) “Lessons from Minamata mercury pollution, Japan – after a continuous 22 years of observation” Wat. Sci. Tech. 38, 187-193.

43. Guallar, M. D., Sanz-Gallardo, M. I., van’t Veer, P., Bode, P., Aro, A., Gómez-Aracena, J., Kark, J. D., Riemersma, R. A., Martín-Moreno, J. M., and Kok, F. J. (2002) “Mercury, Fish Oils, and the risk of Myocardial Infarction” N. Engl. J. Med. 347, 1747-1754.

44. Yoshizawa, K., Rimm, E. B., Morris, J. S., Grandjean, P., Weihe, P., White, R. F., Debes, F., Araki, S., Yokoyama, K., Murata, K, Sorenson, N., Dahl, R. and Jorgensen P. J. (1997) “Cognicit deficit in 7-year-old children with prenatal exposure to methylmercury” Neurotoxicol. Teratol. 19, 417-428.

45. Davidson, P. W., Myers, G. J., Cox, C., Axtell, C., Shamlaye, C., Sloane-Reeves, J. Cernichiari, E., Needham, L, Choi, A., Wang, Y., Berlin, M., and Clarkson, T. W. (1998) “Effects of prenatal and postnatal methylmercury exposure from fish consumption on neurodevelopment; outcomes at sixty-six months of age in the Seychelles child development study” JAMA, 280, 701-707.

46. Clarkson, T. W. and Strain, J. J. (2003) “Nutritional Factors May Modify the Toxic Action of Methyl Mercury in Fish-Eating Populations” J. Nutrition 1539S-1543S.

47. Passos, C. J., Mergler, D., Gaspar, E., Morais, S., Lucotte, M., Larribe, F., Davidson, R., and de Grosbois, S. (2003) “Eating tropical fruit reduces mercury exposure from fish consumption in the Brazilian Amazon” Env. Res. 93, 123-130.

48. Jacobson, J. L. (2001) “Contending with Contradictory Data in a Risk Assessment Context: The Case of Methylmercury” Neurotoxicol. 22, 667-675.

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50. Schell, L. M., Hubicki, L. A., DeCaprio, A. P., Gallo, M. V., Ravenscroft, J., Tarbell, A., Jacobs, A., David, D., Worswick, P. and the Akwesasne Task Force on the Environment. (2003) “Organochlorines, Lead, and Mercury in Akwesasne Mohawk Youth” Env. Health. Persp. 111, 954-961.

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52. Wright, J. G., Tsang, H-T., Penner-Hahn, J. E. and O’Halloran, T. V. (1990) “Coordination Chemistry of the Hg-MerR Metalloregulatory Protein: Evidence for a Novel Tridentate Hg-Cysteine Receptor Site.” J. Am. Chem. Soc. 112, 2434-2435.

53. Zeng, Q., Stålhandske, C., Anderson, M. C., Scott, R. A., and Summers, A. O. (1998) “The Core Metal-Recognition Domain of MerR” Biochemistry, 37, 15885-15895.

Hg SC

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Figure 1: Flexibility of the coordination chemistry of mercury illustrated using computed structures for the methylmercury thiomethane complexes with one, two and three sulfurs coodinating: aCH3HgSCH3, b [CH3Hg(SCH3)2]- and c [CH3Hg(SCH3)3]2-. All unlabelled atoms are hydrogens. All three species are predicted to be quite stable, as will their cysteine analogues.

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Figure 2: Comparison of spectra from samples of concentration (a) 6 µM and (b) 1 µM (200 ppb). Despite the fact that only four spectra were averaged b still shows acceptable signal to noise. Significant improvement will be achieved by signal averaging.

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Fourier transform

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Figure 4: Regions of the X-ray absorption spectrum. The total spectrum (A) can be divided up into the near-edge region (B), and the extended X-ray absorption fine structure (A, inset). This is usually represented in k-space (a measure of the photoelectron kinetic energy), and k-weighted (C), and Fourier transformed (D) to provide a radial structure. Note that the Fourier transform is not generally analyzed directly, but rather the EXAFS oscillations (C) are fitted using a theoretical model. This example shows the data for mercuric-bisthiophenolate. The near-edge spectrum of mercuric oxide (a digonal HgO species) is compared in B (broken line) to illustrate the chemical sensitivity.

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Figure 5: Sensitivity of Hg LIII near-edge spectra to chemical environment - a compares digonallycoordinated oxygen (solid line) and sulfur (broken line) species, b compares HgCl2 (solid line) and [HgCl4]2- (broken line), and c compares digonal Hg(SR)2 (solid line) and [Hg(SR)4]2- (broken line) species. All species are in aqueous solutions.

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Figure 6: EXAFS Fourier transform of preliminary EXAFS data of kidney of a rabbit treated with thimerosal (10mg/kg into the ear vein under anesthesia). The broken line shows the Fourier transform of data from a buffered solution of thimerosal (structure shown in inset) clearly showing Hg-C and Hg-S interactions. Biotransformation of the thimerosal in rabbit kidney is apparent.

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Figure 7: An example of chemically-resolved XAS Imaging (unrelated to the present proposal). The images are of the gameteophyte generation of the fern Pteris vittata which accumulates arsenate from the soil, converting it into arsenite. The intact plant (a) (imaged region highlighted), b photomicrograph, c arsenite, and d arsenate.

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Figure 8: Hg L-edge XAS Imaging of 4-day old zebrafish treated with 100µM methylmercurycysteine for one hour – a shows a photomicrograph, and b and c show X-ray Lα1 fluorescence and transmittance X-ray images, respectively. The swim bladder (sb) is clearly visible in a, b and c. A second orientation of a similarly treated fish is shown in d-f . The mercury can be seen to be concentrated in the eye-balls (e), and in the gastrointenstinal system (b). The embryonic ottoliths can be seen in c and in f. These images were collected using our imaging apparatus on SSRL beamline 9-3 in collaboration with Prof. Pat Krone, University of Saskatchewan.

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Figure 9: Comparison of the Hg LIII near-edge spectrum of swordfish skeletal muscle with a series of model compounds. The points show the spectrum of the fish, while the solid lines show the spectra of aqueous solutions of standard species – (a) CH3HgS(Cys), (b) CH3HgCl, (c) Hg2+( Hg(NO3)2solution), (d) Hg(SR)2, (e) [Hg(SR)4]2-.

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Figure 10: Computed structures of the two possible diasteriomers of the reaction product between stoichiometric meso-dimercaptosuccinic acid (DMSA) and Hg2+ in aqueous solution. The unlabeled atoms are hydrogens.

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Figure 11: Computed energy dependencies of the various bonding parameters of the simple coordination compound CH3SHgSCH3.

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Figure 12: Candidate custom chelator molecule. Note that this is an example only, and not an optimally designed molecule, as the side chains that bind mercury are flexible (ideally these would be rigid). Any unlabelled atoms are hydrogen.

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