Title: the Molecular Basis for Mercury Toxicity

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 - 2- complexes can be achieved with either two [Hg(SR)2], three [Hg(SR)3] , or four [Hg(SR)4] 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 Research Module, Page 12a (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. Research Module, Page 12b 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

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