Jürgen Gailer

Chronic toxicity of AsIII in mammals: The role of (GS)2AsSe-

Millions of people are currently exposed to a multitude of environmentally persistent toxic metals and metalloid compounds mainly through the ingestion of drinking water and food. Despite the fact that biomonitoring studies have revealed several toxic metals to be present in the bloodstream of the general population, the interpretation of the established blood concentrations with regard to their health relevance continues to be an active research area. To this end, a better understanding of the bioinorganic chemistry of individual metals and metalloid compounds in the mammalian bloodstream could greatly advance the interpretation of the available biomonitoring data. Arsenite represents a case in point, since >100 million people are currently exposed to unsafe levels of inorganic arsenic via drinking water. The elucidation of the underlying biomolecular mechanism(s) of toxicity is therefore of the utmost importance and could involve the antagonistic toxic effect between arsenite and selenite, which was discovered in mammals approximately 70 years ago. After a concise overview of animal studies that aimed to understand this trace element antagonism at the molecular level, the in vivo formation and biliary excretion of the seleno-bis(S-glutathionyl) arsinium ion, (GS)(2)AsSe(-), is introduced as a likely biomolecular basis. Arguments in favor of a critical involvement of (GS)(2)AsSe(-) in the chronic toxicity and carcinogenicity of arsenite are presented. The in vivo formation of this toxicologically relevant metabolite in the mammalian bloodstream (mediated by erythrocytes) indicates that the elucidation of bioinorganic chemistry-related mechanisms that take place in the bloodstream represents a promising research strategy to better understand the etiology of numerous human diseases some of which may be ultimately caused by the low level exposure to certain inorganic pollutants.

Analysis of the plasma metalloproteome by SEC–ICP-AES: bridging proteomics and metabolomics

Although blood plasma inherently contains protein biomarkers for human disease diagnosis, their determination is difficult since more than 3700 proteins are commonly present. The associated protein-separation problem can, however, be dramatically simplified by analyzing plasma for a subproteome, such as those proteins that contain bound metals. To this end, the analysis of plasma by size-exclusion chromatography (SEC) coupled with an inductively coupled plasma atomic-emission spectrometer (ICP-AES), which served as the simultaneous Cu-, Fe- and Zn-specific detector, revealed the presence of approximately 12 metalloproteins within 25 min. In the context of modern proteomics research, SEC–ICP-AES therefore represents a viable proteomic approach that can be applied to diagnose human diseases that are associated with increased or decreased concentrations of certain plasma metalloproteins. Furthermore, SEC–ICP-AES can be employed to probe the effect of environmental chemicals or drugs in blood at the metalloprotein level, which makes it a versatile research tool for applications in toxicology, applied medicine, pharmacology and nutritional science.

The separation of dimethylarsinic acid, methylarsonous acid, methylarsonic acid, arsenate and dimethylarsinous acid on the Hamilton PRP-X100 anion-exchange column

In order to separate the potential arsenite metabolites methylarsonous acid and dimethylarsinous acid from arsenite, arsenate, methylarsonic acid and dimethylarsinic acid, the pH-dependent retention behaviour of all six arsenic compounds was studied on a Hamilton PRP-X100 anion-exchange column with 30 mM phosphate buffers (pH 5, 6, 7, 8 and 9) containing 20% (v/v) methanol as mobile phase and employing an inductively coupled plasma atomic emission spectrometer (ICP–AES) as the arsenic-specific detector. Baseline separation of dimethylarsinic acid, methylarsonous acid, methylarsonic acid, arsenate and dimethylarsinous acid was achieved with a 30 mmol dm−3 phosphate buffer (pH 5)–methanol mixture (80:20, v/v) in 25 min. Arsenite is not baseline-separated from dimethylarsinic acid under these conditions. Copyright

How is Inorganic Arsenic Detoxified

Publisher Summary Arsenate is reduced to arsenite enzymatically by arsenate reductase and nonenzymatically by GSH. An early step in the detoxification appears to be the formation of the Gailer compound, seleno-bis(S-glutathionyl) arsinium ion, which is rapidly formed and excreted in the bile. Arsenite-binding proteins initially may prevent or enhance the accumulation of toxic levels of arsenite. As these binding sites become saturated, the arsenite may be released for methylation, a biotransformation process that results in the increase of urinary arsenic (As). Methylation of As species can occur via SAM and methyltransferases and/or nonenzymatically with methylvitamin B 12 , GSH, and selenite. Methylation by the methylvitamin B 12 system has been shown in vitro only. The substrate for DMA production appears to be MMA III . The lack of methyltransferases in many primates strongly indicates that methylation may not be the primary detoxification pathway for Asi. In fact, the environmental protection agency classifies dimethylarsinic acid, the final urinary metabolite for As in humans, as a probable human carcinogen. The determination of the amino acid sequences of the As methyltransferases needs to be accomplished so that gene probes can be constructed to better study As methyltransferase polymorphism, as it is related to the various responses of people to Asi.

Probing the coordination behavior of Hg2+, CH3Hg+, and Cd2+ towards mixtures of two biological thiols by HPLC-ICP-AES

Hepatocyte cytosol contains a multitude of proteins, but also comparatively high concentrations of l-glutathione (GSH, ~5.0 mM) and L-cysteine (Cys, ~0.5 mM). Since Hg(2+), CH(3)Hg(+) and Cd(2+) have a high affinity for thiols, their coordination to these thiols is likely involved in their intracellular transport. The comparative coordination behavior of these metal species towards mixtures of Cys and GSH, however, has not been studied under near physiological conditions. To probe these toxicologically relevant interactions, each metal species was separately injected onto a C(18)-HPLC column (37°C) that had been equilibrated with phosphate buffered saline (PBS) that contained 5.0 mM GSH (mobile phase) and detected with an inductively coupled plasma atomic emission spectrometer. The incremental increase of the Cys concentration in the mobile phase (in 0.5 or 1.0 mM steps) up to 10mM followed by the chromatography of each metal species decreased the retention of Hg(2+) and CH(3)Hg(+) albeit in a different manner. This behavior was rationalized in terms of the replacement of hydrophobic GS-moieties coordinated to each mercurial by less hydrophobic Cys-moieties. In contrast, a Cd-peak eluted close to the void volume with all investigated mobile phases. Using X-ray absorption spectroscopy, the Cd-compound that eluted with a PBS-buffer that contained 5.0 mM GSH was structurally characterized as tetrahedral (GS)(4)Cd. Thus, the in vivo formation of (GS)(4)Cd must be considered and HPLC-ICP-AES is identified as a useful tool to probe dynamic bioinorganic processes which involve the interaction of a metal ion with multiple ligands under physiologically relevant conditions.

A possible molecular link between the toxicological effects of arsenic, selenium and methylmercury: methylmercury(II) seleno bis(S-glutathionyl) arsenic(III)

Using a combination of As and Se K-edge and Hg LIII-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.

Methylated Trivalent Arsenic-Glutathione Complexes are More Stable than their Arsenite Analog

The trivalent arsenic glutathione complexes arsenic triglutathione, methylarsonous diglutathione, and dimethylarsinous glutathione are key intermediates in the mammalian metabolism of arsenite and possibly represent the arsenic species that are transported from the liver to the kidney for urinary excretion. Despite this, the comparative stability of the arsenic-sulfur bonds in these complexes has not been investigated under physiological conditions resembling hepatocyte cytosol. Using size-exclusion chromatography and a glutathione-containing phosphate buffered saline mobile phase (5 or 10 mM glutathione, pH 7.4) in conjunction with an arsenic-specific detector, we chromatographed arsenite, monomethylarsonous acid, and dimethylarsinous acid. The on-column formation of the corresponding arsenic-glutathione complexes between 4 and 37°C revealed that methylated arsenic-glutathione complexes are more stable than arsenic triglutathione. The relevance of these results with regard to the metabolic fate of arsenite in mammals is discussed.

Synthesis, X-ray absorption spectroscopy and purification of the seleno-bis (S-glutathionyl) arsinium anion from selenide, arsenite and glutathione

We report a new synthesis of the seleno-bis (S-glutathionyl) arsinium anion, [(GS)2AsSe]−. An aqueous solution of bis-glutathionylarsenous acid, (GS)2AsOH, prepared from stoichiometric glutathione and arsenite, was reacted in situ with a solution of sodium hydrogen selenide, prepared from elemental selenium and sodium borohydride. Analysis of the arsenic and selenium K-edge X-ray absorption spectra indicated virtually quantitative formation of [(GS)2AsSe]−, with AsSe and AsS distances of 2.31 and 2.25 A, respectively, and the concentrated sample allowed a definitive X-ray spectroscopic characterization. Size-exclusion chromatography was used to separate [(GS)2AsSe]− from residual borate in the reaction mixture.

Probing the bioinorganic chemistry of toxic metals in the mammalian bloodstream to advance human health

The etiology of numerous grievous human diseases, including Alzheimers and Parkinsons Disease is not well understood. Conversely, the concentration toxic metals and metalloids, such as As, Cd, Hg and Pb in human blood of the average population is well established, yet we know strikingly little about the role that they might play in the etiology of disease processes. Establishing functional connections between the chronic exposure of humans to these and other inorganic pollutants and the etiology of certain human diseases is therefore viewed by many as one of the greatest challenges in the post-genomic era. Conceptually, this task requires us to uncover hitherto unknown biomolecular mechanisms which must explain how small doses of a toxic metal/metalloid compound (low μg per day) - or mixtures thereof - may eventually result in a particular human disease. The biological complexity that is inherently associated with mammals, however, makes the discovery of these mechanisms a truly monumental task. Recent findings suggest that a better understanding of the bioinorganic chemistry of inorganic pollutants in the mammalian bloodstream represents a fruitful strategy to unravel relevant biomolecular mechanisms. The adverse effect(s) that toxic metals/metalloid compounds exert on the transport of essential ultratrace elements to internal organs appear particularly pertinent. A brief overview of the effect that arsenite and Hg(2+) exert on the mammalian metabolism of selenium is presented.

Probing the interaction of arsenobetaine with blood plasma constituents in vitro: an SEC-ICP-AES study

Arsenobetaine, which is frequently ingested by humans via the consumption of seafood, is rapidly excreted unchanged in urine, but not much is known about its transport in the mammalian bloodstream. To assess whether this transport involves binding to plasma proteins, rabbit and human plasma were spiked with arsenobetaine and the mixture was analyzed (after 5 min and again after 6 h) by size-exclusion chromatography (SEC) coupled on-line to an inductively coupled plasma atomic emission spectrometer (ICP-AES). Simultaneous monitoring of the emission lines of As, Cu, Fe and Zn in the column effluent allowed us to determine the elution of arsenobetaine relative to that of the major Cu, Fe and Zn-containing metalloproteins. Over the investigated time period, a single As peak eluted near the inclusion volume on two different SEC columns with fractionation ranges of 600-10 KDa and 7000-100 Da. These results indicate that arsenobetaine did not bind to plasma proteins and that SEC-ICP-AES is a useful tool to rapidly probe toxicologically and pharmacologically-relevant interactions between organometalloid compounds and mammalian blood plasma constituents in vitro.