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Mercury is ubiquitous in the environment, with rising levels due to pollution and climate change being a current global concern. Many mercury compounds are notorious for their toxicity, with the potential of organometallic mercury compounds for devastating effects on the structures and functions of the central nervous system being of particular concern. Chronic exposure of human populations to low levels of methylmercury compounds occurs through consumption of fish and other seafood, although the health consequences, if any, from this exposure remain controversial. We have used high energy resolution fluorescence detected X-ray absorption spectroscopy (HERFD-XAS) to determine the speciation of mercury and selenium in human brain tissue. We show that the molecular fate of mercury differs dramatically between individuals who suffered acute organometallic mercury exposure (poisoning) and individuals with chronic low-level exposure from a diet rich in marine fish. For long-term low-level methylmercury exposure from fish consumption, mercury speciation in brain tissue shows methylmercury coordinated to an aliphatic thiolate, resembling the coordination environment observed in marine fish. In marked contrast, for short-term high-level exposure we observe the presence of biologically less-available mercuric selenide deposits, confirmed by X-ray fluorescence imaging, as well as mercury(II)-bis-thiolate complexes, which may be signatures of severe poisoning in humans. These differences between low-level and high-level exposures challenge the relevance of studies involving acute exposure as a proxy for low-level chronic exposure.

Neuronal and sensory toxicity of mercury (Hg) compounds has been largely investigated in humans/mammals with a focus on public health, while research in fish is less prolific and dispersed by different species. Well-established premises for mammals have been governing fish research, but some contradictory findings suggest that knowledge translation between these animal groups needs prudence [e.g. the relative higher neurotoxicity of methylmercury (MeHg) vs. inorganic Hg (iHg)]. Biochemical/physiological differences between the groups (e.g. higher brain regeneration in fish) may determine distinct patterns. This review undertakes the challenge of identifying sensitive cellular targets, Hg-driven biochemical/physiological vulnerabilities in fish, while discriminating specificities for Hg forms., Thanks are due for the financial support to CESAM (UID/AMB/50017/2019), to FCT/MEC through national funds, and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020. Patrícia Pereira acknowledges the support of FCT through the post-doctoral research grants (SFRH/BPD/69563/2010 and SFRH/BPD/107718/2015). Currently, Patrícia Pereira is funded by national funds (OE), through FCT – Fundação para a Ciência e a Tecnologia, I.P., in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19. The authors are also grateful to the Co-Editors Professor Cristina Carvalho and Professor Michael Aschner for the opportunity to publish this article in a special issue of BBAGEN organized under their supervision.

Melano-macrophage aggregates, collections of specialized cells of the innate immune system of fish, are considered a general biomarker for contaminant toxicity. To elucidate further the relationship between macrophage aggregates and metals exposure, yelloweye rockfish (Sebastes ruberrimus), a long-lived species, were sampled from the east and west coasts of Prince of Wales Island, Alaska. Metals concentrations in livers (inorganic Hg, methyl mercury, Se, Ni, Cd, Cu, Zn) and spleens (inorganic Hg and methyl mercury) were determined, as well as their correlations with melano-macrophage aggregate area. Sections of liver tissue were analyzed by laser ablation-inductively coupled plasma-mass spectrometry to determine how metals were spatially distributed between hepatocytes and macrophage aggregates. The concentration of inorganic Hg in whole tissue was the best predictor of macrophage area in yelloweye livers and spleens. Macrophage aggregates had higher relative concentrations than most metals compared with the surrounding hepatocytes. However, not all metals were accumulated to the same degree, as evidenced by differences in the ratios of metals in macrophages compared with hepatocytes. Laser ablation data were corroborated with the results of X-ray synchrotron fluorescence imaging of a yelloweye liver section. Hepatic macrophage aggregates in yelloweye rockfish may play an important role in the detoxification and storage of Hg and other metals.

Methylmercury (MeHg) is a ubiquitous and persistent neurotoxin that poses a risk to human health. Although the mechanisms of MeHg toxicity are not fully understood, factors that contribute to susceptibility are even less well known. Studies of human gene polymorphisms have identified a potential role for the multidrug resistance-like protein (MRP/ABCC) family, ATP-dependent transporters, in MeHg susceptibility. MRP transporters have been shown to be important for MeHg excretion in adult mouse models, but their role in moderating MeHg toxicity during development has not been explored. We therefore investigated effects of manipulating expression levels of MRP using a Drosophila development assay. Drosophila MRP (dMRP) is homologous to human MRP1–4 (ABCC1–4), sharing 50% identity and 67% similarity with MRP1. A greater susceptibility to MeHg is seen in dMRP mutant flies, demonstrated by reduced rates of eclosion on MeHg-containing food. Furthermore, targeted knockdown of dMRP expression using GAL4>UAS RNAi methods demonstrates a tissue-specific function for dMRP in gut, Malpighian tubules, and the nervous system in moderating developmental susceptibility to MeHg. Using X-ray synchrotron fluorescence imaging, these same tissues were also identified as the highest Hg-accumulating tissues in fly larvae. Moreover, higher levels of Hg are seen in dMRP mutant larvae compared with a control strain fed an equivalent dose of MeHg. In sum, these data demonstrate that dMRP expression, both globally and within Hg-targeted organs, has a profound effect on susceptibility to MeHg in developing flies. Our findings point to a potentially novel and specific role for dMRP in neurons in the protection against MeHg. Finally, this experimental system provides a tractable model to evaluate human polymorphic variants of MRP and other gene variants relevant to genetic studies of mercury-exposed populations.

From the perspective of a chemist, biology confers a rich variety of roles on a number of metal ions. It is widely agreed that a large fraction of the genomic output of living things contains metal or metalloid ions, although estimates of this fraction vary widely and depend upon which metal ions are considered.1−3 Moreover, recent reports suggest that, at least in some cases, there are many uncharacterized metalloproteins.4 With inclusion of the s-block metals such as Na, K, Mg, and Ca, the proportion likely approaches 100%; recent estimates from the protein data bank indicate that the prevalence of heavier metal ions of atomic number above 20 within proteins is around 22%,5 with Zn2+ proteins alone constituting about 11%. Living organisms have an inherent and very rich physical structure, with relevant length scales ranging from the nanometer scale for subcellular structure to hundreds of micrometers and above for tissue, organ, or organism-level organization. The ability to derive the spatial distribution of elements on this diversity of length scales is a key to understanding their function. Metals play essential and central roles in the most important and chemically challenging processes required for life, with active site structures and mechanisms that, at the time of their discovery, have usually not yet been duplicated in the chemical laboratory. Furthermore, diseases of metal dysregulation can cause disruption in the distribution of metals.6 For example, Menke’s disease and Occipital Horn Syndrome,7 and Wilson’s disease,8 involve disruption in copper uptake and excretion, respectively, through mutation in the ATP7A and ATP7B Cu transporters.9 The mechanisms of action of toxic elements such as mercury and arsenic are also of interest, as are essential nonmetal trace elements, such as selenium. Likewise, an increasing number of pharmaceuticals include metals or heavier elements; such chemotherapeutic drugs include the platinum derivatives cisplatin and carboplatin,10 some promising new ruthenium drugs,11 and arsenic trioxide, which has been used to treat promyelocytic leukemia.12 Understanding the localization, speciation, and distribution of these at various length scales is of significant interest. A wide variety of heavier elements can be probed by X-ray spectroscopic methods; these are shown graphically in Figure ​Figure1.1. X-ray fluorescence imaging is a powerful technique that can be used to determine elemental and chemical species distributions at a range of spatial resolutions within samples of biological tissues. Most modern applications require the use of synchrotron radiation as a tunable and high spectral brightness source of X-rays. The method uses a microfocused X-ray beam to excite X-ray fluorescence from specific elements within a sample. Because the method depends upon atomic physics, it is highly specific and enables a wide range of chemical elements to be investigated. A significant advantage over more conventional methods is the ability to measure intact biological samples without significant treatment with exogenous reagents. The technique is capable of determining metal and nonmetal distributions on a variety of length scales, with information on chemical speciation also potentially available. Figure ​Figure22 shows examples of rapid-scan X-ray fluorescence imaging at two contrasting length scales: rapid-scan imaging13 of a section of a human brain taken from an individual suffering from multiple sclerosis and showing elemental profiles for Fe, Cu, and Zn;14 and a high-resolution image showing mercury and other elements in a section of retina from a zebrafish larva treated with methylmercury chloride.15 We will discuss both the state of the art in terms of experimental methods and some recent applications of the methods. This Review considers X-ray fluorescence imaging with incident X-ray energies in the hard X-ray regime, which we define as 2 keV and above. We review technologies for producing microfocused X-ray beams and for detecting X-ray fluorescence, as well as methods that confer chemical selectivity or three-dimensional visualization. We discuss applications in key areas with a view to providing examples of how the technique can provide information on biological systems. We also discuss synergy with other methods, which have overlapping or complementary capabilities. Our goal is to provide useful and pertinent information to encourage and enable further use of this powerful method in chemical and biochemical studies of living organisms. Figure 1 Periodic table of the elements showing elements of biological interest that can be probed using X-ray fluorescence imaging. Elements are divided into three categories, those that are physiologically important, those that are pharmacologically active, ...

Human populations experience widespread low level exposure to organometallic methylmercury compounds through consumption of fish and other seafood. At higher levels, methylmercury compounds specifically target nervous systems, and among the many effects of their exposure are visual disturbances, including blindness, which previously were thought to be due to methylmercury-induced damage to the visual cortex. Here, we employ high-resolution X-ray fluorescence imaging using beam sizes of 500 × 500 and 250 × 250 nm(2) to investigate the localization of mercury at unprecedented resolution in sections of zebrafish larvae ( Danio rerio ), a model developing vertebrate. We demonstrate that methylmercury specifically targets the outer segments of photoreceptor cells in both the retina and pineal gland. Methylmercury distribution in both tissues was correlated with that of sulfur, which, together with methylmercury's affinity for thiolate donors, suggests involvement of protein cysteine residues in methylmercury binding. In contrast, in the lens, the mercury distribution was different from that of sulfur, with methylmercury specifically accumulating in the secondary fiber cells immediately underlying the lens epithelial cells rather than in the lens epithelial cells themselves. Since methylmercury targets two main eye tissues (lens and photoreceptors) that are directly involved in visual perception, it now seems likely that the visual disruption associated with methylmercury exposure in higher animals including humans may arise from direct damage to photoreceptors, in addition to injury of the visual cortex.

Fusarium head blight (FHB) is a serious disease of wheat worldwide. Cultivar resistance to FHB depends on biochemical factors that confine the pathogen spread in spikes. Breeding for cultivar resistance is considered the most practical way to manage this disease. In this study, different spectroscopy and microscopy techniques were applied to discriminate resistance in wheat genotypes against FHB. Synchrotron-based spectroscopy and imaging techniques, including focal plane array infrared and X-ray fluorescence (XRF) spectroscopy were used to understand changes in biochemical and trace elements in rachis following FHB infection. Sumai3 and Muchmore were used to represent resistant and susceptible cultivars to FHB, respectively, in this study. The histological comparison of rachis showed substantial differences in the cell wall thickness between the cultivars after infection. Synchrotron-based infrared imaging emphasized substantial difference in biochemical composition of rachis samples between the two cultivars prior to visible symptoms; in the resistant Sumai3, infrared bands representing alkyl ester, cellulose and lignin vibrations as well as amide I, pectin, cellulose, hemicellulose, and aromatics group were stronger and more persistent compared to the susceptible cultivar. These bands may be the candidates of biochemical markers for FHB resistance. Focal plane array infrared imaging (FPA) spectra from the rachis epidermis and vascular bundles revealed a new band (1710 cm-1) related to the oxidative stress on the susceptible cultivar only. XRF spectroscopy data revealed differences in trace elemental composition between cultivars, and between non-inoculated and inoculated samples, with substantial increases observed for Ca, K, Mn, Fe, Zn, and Si in the resistant cultivar. These trace elements are related to cell wall stability, metabolic process, and plant defense mechanisms such as lignification pathway and callose deposition. The combination of cell wall elemental composition and lignification plays a role in the mechanism of type II host resistance to FHB. Biochemical profiling using the synchrotron-based spectroscopy holds potential for screening wheat genotypes for FHB resistance.

Mercury, one of the most toxic elements, exists in various chemical forms each with different toxicities and health implications. Some methylated mercury forms, one of which exists in fish and other seafood products, pose a potential threat, especially during embryonic and early postnatal development. Despite global concerns, little is known about the mechanisms underlying transport and toxicity of different mercury species. To investigate the impact of different mercury chemical forms on vertebrate development, we have successfully combined the zebrafish, a well-established developmental biology model system, with synchrotron-based X-ray fluorescence imaging. Our work revealed substantial differences in tissue-specific accumulation patterns of mercury in zebrafish larvae exposed to four different mercury formulations in water. Methylmercury species not only resulted in overall higher mercury burdens but also targeted different cells and tissues than their inorganic counterparts, thus revealing a significant role of speciation in cellular and molecular targeting and mercury sequestration. For methylmercury species, the highest mercury concentrations were in the eye lens epithelial cells, independent of the formulation ligand (chloride versusl-cysteine). For inorganic mercury species, in absence of l-cysteine, the olfactory epithelium and kidney accumulated the greatest amounts of mercury. However, with l-cysteine present in the treatment solution, mercuric bis-l-cysteineate species dominated the treatment, significantly decreasing uptake. Our results clearly demonstrate that the common differentiation between organic and inorganic mercury is not sufficient to determine the toxicity of various mercury species.

K-Edge Subtraction (KES) utilizes the discontinuity in the X-ray absorption across the absorption edge of the selected contrast element and creates an image of the projected density of the contrast element from two images acquired just above and below the K-edge of the contrast element. KES has proved to be powerful in coronary angiography, micro-angiography, bronchography, and lymphatic imaging. X-ray fluorescence imaging is a successful technique for the detection of dilute quantities of elements in specimens. However, its application at high X-ray energies (e.g. at the iodine K-edge) is complicated by significant Compton background, which may enter the energy window set for the contrast material's fluorescent X-rays. Inspired by KES, Fluorescence Subtraction Imaging (FSI) is a technique for high-energy (>20 keV) fluorescence imaging using two different incident beam energies just above and below the absorption edge of a contrast element (e.g. iodine). The below-edge image can be assumed as a “background” image, which includes Compton scatter and fluorescence from other elements. The above-edge image will contain nearly identical spectral content as the below-edge image but will contain the additional fluorescence of the contrast element. This imaging method is especially promising with thick objects with dilute contrast materials, significant Compton background, and/or competing fluorescence lines from other materials. A quality factor is developed to facilitate the comparison. The theoretical value of the quality factor sets the upper limit that an imaging method can achieve when the noise is Poisson limited. The measured value of this factor makes two or more imaging methods comparable. Using the Hard X-ray Micro-Analysis (HXMA) beamline at the Canadian Light Source (CLS), the techniques of FSI and KES were critically compared, with reference to radiation dose, image acquisition time, resolution, signal-to-noise ratios, and quality factor.

Sulfur is essential for life, with important roles in biological structure and function. However, because of a lack of suitable biophysical techniques, in situ information about sulfur biochemistry is generally difficult to obtain. Here, we present an in situ sulfur X-ray absorption spectroscopy (S-XAS) study of living cell cultures of the mammalian renal epithelial MDCK cell line. A great deal of information is retrieved from a characteristic sulfonate feature in the X-ray absorption spectrum of the cell cultures, which can be related to the amino acid taurine. We followed the time and dose dependence of uptake of taurine into MDCK cell monolayers. The corresponding uptake curves showed a typical saturation behavior with considerable levels of taurine accumulation inside the cells (as much as 40% of total cellular sulfur). We also investigated the polarity of uptake of taurine into MDCK cells, and our results confirmed that uptake in situ is predominantly a function of the basolateral cell surface.

The compounds of mercury can be more toxic than those of any other non-radioactive heavy element. Despite this, environmental mercury pollution and human exposure to mercury are widespread, and are increasing. While the unusual ability of selenium to cancel the toxicity of mercury compounds has been known for nearly five decades, only recently have some aspects of the molecular mechanisms begun to be understood. We report herein a study of the interaction of mercury and selenium in the larval stage zebrafish, a model vertebrate system, using X-ray fluorescence imaging. Exposure of larval zebrafish to inorganic mercury shows nano-scale structures containing co-localized mercury and selenium. No such co-localization is seen with methylmercury exposure under similar conditions. Micro X-ray absorption spectra support the hypothesis that the co-localized deposits are most likely comprised of highly insoluble mixed chalcogenide HgSxSe(1−x) where x is 0.4–0.9, probably with the cubic zincblende structure.

In recent years larval stage zebrafish have been emerging as a standard vertebrate model in a number of fields, ranging from developmental biology to pharmacology and toxicology. The tyrosinase inhibitor 1-phenyl-2-thiourea (PTU) is used very widely with larval zebrafish to generate essentially transparent organisms through inhibition of melanogenesis, which has enabled many elegant studies in areas ranging from neurological development to cancer research. Here we show that PTU can have dramatic synergistic and antagonistic effects on the chemical toxicology of different mercury compounds. Our results indicate that extreme caution should be used when employing PTU in toxicological studies, particularly when studying toxic metal ions.

The iron-sulfur cluster-free hydrogenase (Hmd) from methanogenic archaea harbors an iron-containing cofactor of yet unknown structure. X-ray absorption spectroscopy of the active, as isolated enzyme from Methanothermobacter marburgensis (mHmd) and of the active, reconstituted enzyme from Methanocaldococcus jannaschii (jHmd) revealed the presence of mononuclear iron with two CO, one sulfur and one or two N/O in coordination distance. In jHmd, the single sulfur ligand is most probably provided by Cys176, as deduced from a comparison of the activity and of the x-ray absorption and Mössbauer spectra of the enzyme mutated in any of the three conserved cysteines. In the isolated Hmd cofactor, two CO, one sulfur, and two nitrogen/oxygen atoms coordinate the iron, the sulfur ligand being most probably provided by mercaptoethanol, which is absolutely required for the extraction of the iron-containing cofactor from the holoenzyme and for the stabilization of the extracted cofactor. In active mHmd holoenzyme, the number of iron ligands increased by one when one of the Hmd inhibitors (CO or KCN) were present, indicating that in active Hmd, the iron contains an open coordination site, which is proposed to be the site of H2 interaction.

Calcification of the aortic valve cusps due to degenerative changes is the most common lesion encountered in patients who require aortic valve replacement. The primary pathogenic mechanisms responsible for calcific degeneration of the aortic valves still remain unclear. Moreover, there are no convenient methods for the assessment of aortic valve susceptibility to mineralization, which is of crucial importance for the choice of valves for transplantation. The present study was undertaken to develop a quick method for the assessment of the mineralization of aortic valve samples by determination of the increase in calcium level during in vitro incubation of aorta samples. Such a method would allow one to obtain the necessary information without destruction of the material for transplantation. The study consisted in the incubation of human aorta and aortic valve samples, followed by the determination of calcium concentrations by EDXRF. The following elements were detected: Na, Mg, Al, Si, P, S, Cl, K, Fe, Ni, Cu, Zn and Br. Compton scattering was used for determination of the sample thickness. The results showed that if there is an increase in Ca level in aortic valves it is accompanied by its elevation in aorta. The study showed that EDXRF may be a useful tool for studies of mineralization processes in aortic valve and aorta samples.

The influence of gallium ions on the development of the mineralization process in a cell culture model was investigated. To determine the elemental composition the proton microprobe in combination with proton induced X-ray emission (micro-PIXE) and energy-dispersive X-ray fluorescence (XRF) were used. Micro-PIXE allows a precise quantification of elements combined with their accurate localization. In contrast, the XRF results may be used as a rough estimate of the mineralization process development. It was found that gallium influences the mineralization process in a dose-dependent manner. At low doses an increased while at high doses an impaired mineralization was observed. The amount of deposited gallium correlates with the concentration of the element in the cell culture medium. © 2001 Elsevier Science B.V. All rights reserved.

Methylmercury is among the most potentially toxic species to which human populations are exposed, both at high levels through poisonings and at lower levels through consumption of fish and other seafood. However, the molecular mechanisms of methylmercury toxicity in humans remain poorly understood. We used synchrotron X-ray absorption spectroscopy (XAS) to study mercury chemical forms in human brain tissue. Individuals poisoned with high levels of methylmercury species showed elevated cortical selenium with significant proportions of nanoparticulate mercuric selenide plus some inorganic mercury and methylmercury bound to organic sulfur. Individuals with a lifetime of high fish consumption showed much lower levels of mercuric selenide and methylmercury cysteineate. Mercury exposure did not perturb organic selenium levels. These results elucidate a key detoxification pathway in the central nervous system and provide new insights into the appropriate methods for biological monitoring.

Neurotoxic methylmercury compounds are widespread in the environment and human exposure worries many communities worldwide. Despite numerous studies addressing methylmercury toxicity, the detailed mechanisms underlying its transport and accumulation, especially during early developmental stages, remain unclear. Zebrafish larvae are increasingly used as a model system for studies of vertebrate development and toxicology. Previously, we have identified the lens epithelium as the primary site for cellular mercury accumulation in developing zebrafish larvae (Korbas et al. in Proc Natl Acad Sci USA 105:12108–12112, 2008). Here we present a study on the dynamics of methylmercury accumulation and redistribution in the lens following embryonic and larval exposure to methylmercury l-cysteineate using synchrotron X-ray fluorescence imaging. We observed highly specific accumulation of mercury in the lens that continues well after removal of fish from treatment solutions, thus significantly increasing the post-exposure loading of mercury in the lens. The results indicate that mercury is redistributed from the original target tissue to the eye lens, identifying the developing lens as a major sink for methylmercury in early embryonic and larval stages.

The characterization of a novel Mo-Fe protein (MorP) associated with a system that responds to Mo in Desulfovibrio alaskensis is reported. Biochemical characterization shows that MorP is a periplasmic homomultimer of high molecular weight (260 ± 13 kDa) consisting of 16-18 monomers of 15321.1 ± 0.5 Da. The UV/visible absorption spectrum of the as-isolated protein shows absorption peaks around 280, 320, and 570 nm with extinction coefficients of 18700, 12800, and 5000 M-1 cm-1, respectively. Metal content, EXAFS data and DFT calculations support the presence of a Mo-2S-[2Fe-2S]-2S-Mo cluster never reported before. Analysis of the available genomes from Desulfovibrio species shows that the MorP encoding gene is located downstream of a sensor and a regulator gene. This type of gene arrangement, called two component system, is used by the cell to regulate diverse physiological processes in response to changes in environmental conditions. Increase of both gene expression and protein production was observed when cells were cultured in the presence of 45 μM molybdenum. Involvement of this system in Mo tolerance of sulfate reducing bacteria is proposed. Fil: Rivas, Maria Gabriela. Requimte/cqfb, Departamento de Quimica, Fct-unl; Portugal Fil: Carepo, Marta S.P.. Requimte/cqfb, Departamento de Quimica, Fct-unl; Portugal Fil: Mota, Cristiano S.. Requimte/cqfb, Departamento de Quimica, Fct-unl; Portugal Fil: Korbas, Malgorzata. University of Saskatchewan; Canadá Fil: Durand, Marie-Claire. Cnrs-imm, Unité Interactions Et Modulateurs de Réponses; Francia Fil: Lopes, Ana T.. Requimte/cqfb, Departamento de Quimica, Fct-unl; Portugal Fil: Brondino, Carlos Dante. Universidad Nacional del Litoral; Argentina Fil: Pereira, Alice S.. Requimte/cqfb, Departamento de Quimica, Fct-unl; Portugal Fil: George, Graham N.. University of Saskatchewan; Canadá Fil: Dolla, Alain. Cnrs-imm, Unité Interactions Et Modulateurs de Réponses; Francia Fil: Moura, José J.G.. Requimte/cqfb, Departamento de Quimica, Fct-unl; Portugal Fil: Moura, Isabel. Requimte/cqfb, Departamento de Quimica, Fct-unl; Portugal

Using synchrotron x-ray fluorescence mapping, we have examined the uptake and localization of organic mercury in zebrafish larvae. Strikingly, the greatest accumulation of methyl and ethyl mercury compounds was highly localized in the rapidly dividing lens epithelium, with lower levels going to brain, optic nerve, and various other organs. The data suggest that the reported impairment of visual processes by mercury may arise not only from previously reported neurological effects, but also from direct effects on the ocular tissue. This novel approach is a powerful tool for directly investigating the molecular toxicology of heavy metals, and should be equally applicable to the study of a wide range of elements in developing embryos.

Publisher Summary This chapter highlights bioinorganic chemistry underlying the molecular toxicology of heavy metals and metalloids, with particular reference to mercury and arsenic. It is not intended to be a comprehensive review of the entire field of bioinorganic molecular toxicology, but instead, it provides a focused examination of selected areas to illustrate the application of the in situ spectroscopic technique of X-ray absorption spectroscopy (XAS), and the use of quantum mechanical molecular modeling in toxicology. XAS is a powerful tool for investigation of both physical and electronic structure. The major strength of XAS is that it can be used to obtain molecular information on a sample with essentially no pre-treatment. X-ray absorption spectra arise from core-level excitation by absorption of X-rays, and are thus associated with an absorption edge. The increasing availability and sensitivity of in situ advanced spectroscopic probes such as XAS is expected to support important advances in molecular toxicology. The use of advanced quantum mechanical codes in combination with in situ spectroscopic probes has significant advantages in providing a reality check. Design of custom chelators is expected to be important in therapy of heavy metal poisoning. The specificity of such chelation drugs can be especially important as exemplified by recent deaths from hypocalcemia with ethylenediaminetetraacetic acid (EDTA) chelation therapy.

Using a combination of As and Se K-edge and Hg L(III)-edge X-ray absorption spectroscopy, 77Se nuclear magnetic resonance spectroscopy, electrospray ionization mass spectrometry and molecular modeling, we have structurally characterized the novel species methylmercury(II) seleno bis(S-glutathionyl) arsenic(III). This species is formed in aqueous solution from CH3HgOH and the seleno bis(S-glutathionyl) arsinium ion and constitutes an important first step towards characterizing the observed toxicologically relevant interaction between arsenite, selenite and methylmercury which has been previously reported in mammals.

The Ni-Fe carbon monoxide (CO) dehydrogenase II (CODHII(Ch)) from the anaerobic CO-utilizing hydrogenogenic bacterium Carboxydothermus hydrogenoformans catalyzes the oxidation of CO, presumably at the Ni-(micro(2)S)-Fe1 subsite of the [Ni-4S-5S] cluster in the active site. The CO oxidation mechanism proposed on the basis of several CODHII(Ch) crystal structures involved the apical binding of CO at the nickel ion and the activation of water at the Fe1 ion of the cluster. To understand how CO interacts with the active site, we have studied the reactivity of the cluster with potassium cyanide and analyzed the resulting type of nickel coordination by x-ray absorption spectroscopy. Cyanide acts as a competitive inhibitor of reduced CODHII(Ch) with respect to the substrate CO and is therefore expected to mimic the substrate. It inhibits the enzyme reversibly, forming a nickel cyanide. In this reaction, one of the four square-planar sulfur ligands of nickel is replaced by the carbon atom of cyanide, suggesting removal of the micro(2)S from the Ni-(micro(2)S)-Fe1 subsite. Upon reactivation of the inhibited enzyme, cyanide is released, and the square-planar coordination of nickel by 4S ligands is recovered, which includes the reformation of the Ni-(micro(2)S)-Fe1 bridge. The results are summarized in a model of the CO oxidation mechanism at the [Ni-4Fe-5S] active site cluster of CODHII(Ch) from C. hydrogenoformans.

The oncoprotein E7 of human papilloma viruses (HPV) is involved in the pathogenesis and maintenance of human cervical cancers. The most prevalent HPV types found in cervix carcinomas are HPV16, 18 and 45. The structure of the E7 dimer from HPV45 (PDB 2F8B) was determined by nuclear magnetic resonance spectroscopy. Each monomer comprises an unfolded N-terminus and a well-structured C-terminal domain with a beta1beta2alpha1beta3alpha2 topology representing a unique zinc-binding fold found only for E7. Dimerization occurs through the alpha1/alpha1' helices and intermolecular beta-sheet formation but excludes the zinc-binding sites. E7 is reported to interact with a number of cellular proteins (e.g. pRb, p21(CIP1)). Binding of a peptide derived from the C-terminus of p21(CIP1) to the C-terminal domain of E7 was characterized by monitoring chemical shift perturbations of the amide groups of E7. This provides direct evidence that a shallow groove situated between alpha1 and beta1 of the E7 C-terminal domain is interacting with the C-terminus of p21(CIP1). Intriguingly, this binding site overlaps with the low-affinity binding site on E7 for the C-domain of pRb.

During mineral growth in rat bone-marrow stromal cell cultures, gallium follows calcium pathways. The dominant phase of the cell culture mineral constitutes the poorly crystalline hydroxyapatite (HAP). This model system mimics bone mineralization in vivo. The structural characterization of the Ga environment was performed by X-ray absorption spectroscopy at the Ga K-edge. These data were compared with Ga-doped synthetic compounds (poorly crystalline hydroxyapatite, amorphous calcium phosphate and brushite) and with strontium-treated bone tissue, obtained from the same culture model. It was found that Sr(2+) substitutes for Ca(2+) in the HAP crystal lattice. In contrast, the replacement by Ga(3+) yielded a much more disordered local environment of the probe atom in all investigated cell culture samples. The coordination of Ga ions in the cell culture minerals was similar to that of Ga(3+), substituted for Ca(2+), in the Ga-doped synthetic brushite (Ga-DCPD). The Ga atoms in the Ga-DCPD were coordinated by four oxygen atoms (1.90 A) of the four phosphate groups and two oxygen atoms at 2.02 A. Interestingly, the local environment of Ga in the cell culture minerals was not dependent on the onset of Ga treatment, the Ga concentration in the medium or the age of the mineral. Thus, it was concluded that Ga ions were incorporated into the precursor phase to the HAP mineral. Substitution for Ca(2+ )with Ga(3+) distorted locally this brushite-like environment, which prevented the transformation of the initially deposited phase into the poorly crystalline HAP.

Human exposure to potentially neurotoxic methylmercury species is a public-health concern for many populations worldwide. Both fish and whale are known to contain varying amounts of methylmercury species. However studies of populations that consume large quantities of fish or whale have provided no clear consensus as to the extent of the risk. The toxicological profile of an element depends strongly on its chemical form. We have used X-ray absorption spectroscopy to investigate the comparative chemical forms of mercury and selenium in fish and whale skeletal muscle. The predominant chemical form of mercury in whale is found to closely resemble that found in fish. In the samples of skeletal muscle studied, no involvement of selenium in coordination of mercury is indicated in either whale or fish, with no significant inorganic HgSe or HgS type phases being detected. The selenium speciation in fish and whale shows that similar chemical types are present in each, but in significantly different proportions. Our results suggest that for equal amounts of Hg in skeletal muscle, the direct detrimental effects arising from the mercury content from consuming skeletal muscle from whale and fish should be similar if the effects of interactions with other components in the meat are not considered.

Automation of x-ray absorption spectroscopic (XAS) data reduction is essential to cope with high-throughput data collection becoming available at an increasing number of synchrotron radiation centers. A flexible script called KEMP has been developed and implemented at the XAS beamline at EMBL Hamburg. It automatically processes fluorescence XAS data. The pipeline includes dead time correction, energy calibration, selection of fluorescence detector channels, as well as the extraction of x-ray absorption near-edge structure and extended x-ray-absorption fine structure. The output is quickly available and thus can be included in the design of further experiments, which results in a more efficient use of the beam time.