Interaction of carbonic anhydrase I released from red blood cells with human plasma in vitro.

Red blood cells (RBCs) constitute ∼50% of the bloodstream and represent an important target for environmental pollutants and bacterial/viral infections, which can result in their rupture. In addition, diseases such as sickle cell anaemia and paroxysmal nocturnal haemoglobinuria can also result in the rupture of RBCs, which can be potentially life-threatening. With regard to the release of cytosolic metalloproteins from RBCs into the blood-organ system, the biochemical fate of haemoglobin is rather well understood, while comparatively little is known about another highly abundant Zn-metalloprotein, carbonic anhydrase (CA I). To gain insight into the interaction of CA I with human blood plasma constituents, we have employed a metallomics tool comprised of size-exclusion chromatography (SEC) coupled online with an inductively coupled plasma atomic emission spectrometer (ICP-AES), which allows to simultaneously observe all Cu, Fe, and Zn-metalloproteins. After the addition of CA I to human blood plasma incubated at 37°C, the SEC-ICP-AES analysis using phosphate buffered saline (pH 7.4) after 5 min, 1 h, and 2 h revealed that CA I eluted after all endogenous Zn-metalloproteins in the 30 kDa range. Matrix-assisted laser desorption-time of flight mass spectrometry analysis of the collected Zn-peak confirmed that CA I eluted from the column intact. Our in vitro results suggest that CA I released from RBCs to plasma remains free and may be actively involved in health-relevant adverse processes that unfold at the bloodstream-endothelial interface, including atherosclerosis and vision loss.

Toward a Mechanism-Driven Integrated Framework to Link Human Exposure to Multiple Toxic Metal(loid) Species with Environmental Diseases.

The ongoing anthropogenic pollution of the biosphere with As, Cd, Hg and Pb will inevitably result in an increased influx of their corresponding toxic metal(loid) species into the bloodstream of human populations, including children and pregnant women. To delineate whether the measurable concentrations of these inorganic pollutants in the bloodstream are tolerable or implicated in the onset of environmental diseases urgently requires new insight into their dynamic bioinorganic chemistry in the bloodstream-organ system. Owing to the human exposure to multiple toxic metal(loid) species, the mechanism of chronic toxicity of each of these needs to be integrated into a framework to better define the underlying exposure-disease relationship. Accordingly, this review highlights some recent advances into the bioinorganic chemistry of the Cd2+, Hg2+ and CH3Hg+ in blood plasma, red blood cells and target organs and provides a first glimpse of their emerging mechanisms of chronic toxicity. Although many important knowledge gaps remain, it is essential to design experiments with the intent of refining these mechanisms to eventually establish a framework that may allow us to causally link the cumulative exposure of human populations to multiple toxic metal(loid) species with environmental diseases of unknown etiology that do not appear to have a genetic origin. Thus, researchers from a variety of scientific disciplines need to contribute to this interdisciplinary effort to rationally address this public health threat which may require the implementation of stronger regulatory requirements to improve planetary and human health, which are fundamentally intertwined.

Mercury binding to proteins disclosed by ESI MS experiments: The case of three organomercurials.

Solution interactions of three organomercury compounds, i.e., methylmercury chloride, thimerosal and phenylmercury acetate, with a group of biochemically relevant proteins, namely cytochrome c (Cyt c), ribonuclease A (RNase A), carbonic anhydrase I (hCA I), superoxide dismutase (SOD), and serum albumin (HSA), were investigated using an established ESI MS approach. Temporal analysis of sample aliquots provided insight into the binding kinetics, while comparative analysis of the obtained mass spectra disclosed adduct formation of each mercurial with the tested proteins and the relative abundance of the species. The three organomercurials bind, exclusively and tightly, to free cysteine residues as no binding was observed in the case of proteins lacking such groups. hCA I, SOD and HSA formed distinct mercury adducts, preserving the Hg bound alkyl/aryl ligands; yet, the three organomercurials displayed significant differences in reactivity in relation to their chemical structure. The investigation was then extended to analyze the reactions with the C-terminal dodecapeptide of the enzyme human thioredoxin reductase, which contains a characteristic selenol-thiol moiety: tight Hg binding was observed. Notably, this peptide was able to remove effectively and completely the alkyl/aryl ligands of the three tested organomercurials; this behavior may be relevant to the detoxification mechanism of organomercurials in mammals. Finally, a competition experiment was carried out to establish whether protein bound mercury centers may be displaced by other competing metals. Interestingly, and quite unexpectedly, we observed that a protein bound mercury fragment may be partially displaced from its coordination site in hCA I by the medicinal gold compound auranofin.

Translational toxicology of metal(loid) species: linking their bioinorganic chemistry in the bloodstream to organ damage onset.

The quantification of arsenic, mercury, cadmium and lead in the human bloodstream is routinely used today to assess exposure to these toxic metal(loid)s, but the interpretation of the obtained data in terms of their cumulative health relevance remains problematic. Seemingly unrelated to this, epidemiological studies strongly suggest that the simultaneous chronic exposure to these environmental pollutants is associated with the etiology of autism, type 2 diabetes, irritable bowel disease and other diseases. This from a public health point of view undesirable situation urgently requires research initiatives to establish functional connections between human exposure to multiple toxic metal(loid) species and adverse health effects. One way to establish causal exposure-response relationships is a molecular toxicology approach, which requires one to unravel the biomolecular mechanisms that unfold after individual toxic metal(loid)s enter the bloodstream/organ nexus as these interactions ultimately determine which metabolites impinge on target organs and thus provide mechanistic links to diseases of unknown etiology. In an attempt to underscore the importance of the toxicological chemistry of metal(loid)s in the bloodstream, this review summarizes recent progress into relevant bioinorganic processes that are implicated in the etiology of adverse organ-based health effects and possibly diseases. A better understanding of these bioinorganic processes will not only help to improve the regulatory framework to better protect humans from the adverse effects of toxic metal(loid) species, but also represents an important starting point for the development of treatments to ameliorate pollution-induced adverse health effects on human populations, including pregnant women, the fetus and children.

N-Acetylcysteine Displaces Glutathionyl-Moieties from Hg2+ and MeHg+ to Form More Hydrophobic Complexes at Near-Physiological Conditions.

The anthropogenic release of Hg is associated with an increased human exposure risk. Since Hg2+ and MeHg+ have a high affinity for thiols, their interaction with L-glutathione (GSH) within mammalian cells is fundamentally involved in their toxicological chemistry and excretion. To gain insight into the interaction of these mercurials with multiple small molecular weight thiols, we have investigated their competitive interactions with GSH and N-acetylcysteine (NAC) at near-physiological conditions, using a liquid chromatographic approach. This approach involved the injection of each mercurial onto a reversed-phase (RP)-HPLC column (37 °C) using a PBS buffer mobile phase containing 5.0 mM GSH to simulate cytosolic conditions with Hg being detected in the column effluent by an inductively coupled plasma atomic emission spectrometer (ICP-AES). When the 5.0 mM GSH mobile phase was amended with up to 10 mM NAC, gradually increasing retention times of both mercurials were observed. To explain this behavior, the experiment with 5.0 mM NAC and 5.0 mM GSH was replicated using 50 mM Tris buffer (pH 7.4), and the Hg-containing fractions were analyzed by electrospray ionization mass spectrometry. The results revealed the presence of Hg(GS)(NAC) and Hg(NAC)2 for Hg2+ and MeHg(GS) and MeHg(NAC) for MeHg+, which suggests that the coordination/displacement of GS-moieties from each mercurial by the more hydrophobic NAC can explain their retention behavior. Since the biotransformations of both mercurials were observed at near-physiological conditions, they are of toxicological relevance as they provide a biomolecular explanation for some results that were obtained when animals were administered with each mercurial and NAC.

More Effective Mobilization of Hg2+ from Human Serum Albumin Compared to Cd2+ by L-Cysteine at Near-Physiological Conditions.

Although chronic low-level exposure to Hg2+ and Cd2+ causes human nephrotoxicity, the bioinorganic processes that deliver them to their target organs are poorly understood. Since the plasma protein human serum albumin (HSA) has distinct binding sites for these metal ions, we wanted to gain insight into these translocation processes and have employed size-exclusion chromatography coupled on-line to an inductively coupled plasma atomic emission spectrometer using phosphate-buffered saline mobile phases. When HSA 'labeled' with Hg2+ and Cd2+ (1:0.1:0.1) using 300 µM of L-methionine was analyzed, the co-elution of a single C, S, Cd, and Hg peak was observed, which implied the intact bis-metalated HSA complex. Since human plasma contains small molecular weight thiols and sulfur-containing metabolites, we analyzed the bis-metalated HSA complex with mobile phases containing 50-200 µM of L-cysteine (Cys), D,L-homocysteine (hCys), or glutathione (GSH), which provided insight into the comparative mobilization of each metal from their respective binding sites on HSA. Interestingly, 50 µM Cys, hCys, or GSH mobilized Hg2+ from its HSA binding site but only partially mobilized Cd2+ from its binding site. Since these findings were obtained at conditions simulating near-physiological conditions of plasma, they provide a feasible explanation for the higher 'mobility' of Hg2+ and its concomitant interaction with mammalian target organs compared to Cd2+. Furthermore, 50 µM Cys resulted in the co-elution of similar-sized Hg and Cd species, which provides a biomolecular explanation for the nephrotoxicity of Hg2+ and Cd2+.

Structural Characterization of Toxicologically Relevant Cd2+-L-Cysteine Complexes.

The exposure of humans to Cd exerts adverse human health effects at low chronic exposure doses, but the underlying biomolecular mechanisms are incompletely understood. To gain insight into the toxicologically relevant chemistry of Cd2+ in the bloodstream, we employed an anion-exchange HPLC coupled to a flame atomic absorption spectrometer (FAAS) using a mobile phase of 100 mM NaCl with 5 mM Tris-buffer (pH 7.4) to resemble protein-free blood plasma. The injection of Cd2+ onto this HPLC-FAAS system was associated with the elution of a Cd peak that corresponded to [CdCl3]-/[CdCl4]2- complexes. The addition of 0.1-10 mM L-cysteine (Cys) to the mobile phase significantly affected the retention behavior of Cd2+, which was rationalized by the on-column formation of mixed CdCysxCly complexes. From a toxicological point of view, the results obtained with 0.1 and 0.2 mM Cys were the most relevant because they resembled plasma concentrations. The corresponding Cd-containing (~30 µM) fractions were analyzed by X-ray absorption spectroscopy and revealed an increased sulfur coordination to Cd2+ when the Cys concentration was increased from 0.1 to 0.2 mM. The putative formation of these toxicologically relevant Cd species in blood plasma was implicated in the Cd uptake into target organs and underscores the notion that a better understanding of the metabolism of Cd in the bloodstream is critical to causally link human exposure with organ-based toxicological effects.

Physiologically relevant hCys concentrations mobilize MeHg from rabbit serum albumin to form MeHg-hCys complexes.

Methylmercury (MeHg) is one of the most potent neurotoxins to which humans are exposed via the consumption of fish, from which it is effectively absorbed via the gastrointestinal tract into the bloodstream. Its interactions with plasma proteins, small-molecular-weight (SMW) molecules, and red blood cells, however, are incompletely understood, but critical as they determine whether and how much MeHg reaches target organs. To better define the role that SMW thiols play in the delivery of MeHg to known transporters located at the placental and blood-brain barrier, we have employed size exclusion chromatography-inductively coupled plasma-atomic emission spectroscopy to analyze MeHg-spiked rabbit plasma in the absence and presence of SMW thiols dissolved in the phosphate-buffered saline buffer mobile phase. While 300 µM methionine did not affect the binding of MeHg to rabbit serum albumin (RSA), cysteine (Cys), homocysteine (hCys), and glutathione resulted in the elution of the main Hg peak in the SMW elution range. In addition, 50 µM of hCys or Cys in the mobile phase resulted in the mobilization of MeHg from RSA in rabbit plasma and from pure RSA in solution. The Hg peak that eluted in the SMW elution range (50 µM of hCys) was identified by electrospray ionization-mass spectrometry as an MeHg-hCys complex. Since l-type amino acid transporters are present at the blood-brain barrier (BBB), which facilitate the uptake of MeHg-Cys species into the brain, our results contribute to establish the bioinorganic mechanisms that deliver MeHg to the BBB, which is critical to predict organ-based adverse health effects.

Quantification of human plasma metalloproteins in multiple sclerosis, ischemic stroke and healthy controls reveals an association of haptoglobin-hemoglobin complexes with age.

Advanced analytical methods play an important role in quantifying serum disease biomarkers. The problem of separating thousands of proteins can be reduced by analyzing for a 'sub-proteome', such as the 'metalloproteome', defined as all proteins that contain bound metals. We employed size exclusion chromatography (SEC) coupled to an inductively coupled plasma atomic emission spectrometer (ICP-AES) to analyze plasma from multiple sclerosis (MS) participants (n = 21), acute ischemic stroke (AIS) participants (n = 17) and healthy controls (n = 21) for Fe, Cu and Zn-metalloproteins. Using ANOVA analysis to compare the mean peak areas among the groups revealed no statistically significant differences for ceruloplasmin (p = 0.31), α2macroglobulin (p = 0.51) and transferrin (p = 0.31). However, a statistically significant difference was observed for the haptoglobin-hemoglobin (Hp-Hb) complex (p = 0.04), being driven by the difference between the control group and AIS (p = 0.012), but not with the MS group (p = 0.13), based on Dunnes test. A linear regression model for Hp-Hb complex with the groups now adjusted for age found no statistically significant differences between the groups (p = 0.95), but was suggestive for age (p = 0.057). To measure the strength of association between the Hp-Hb complex and age without possible modifications due to disease, we calculated the Spearman rank correlation in the healthy controls. The latter revealed a positive association (r = 0.39, 95% Confidence Interval = (-0.05, 0.83), which suggests that either the removal of Hp-Hb complexes from the blood circulation slows with age or that the release of Hb from red blood cells increases with age. We also observed that the Fe-peak corresponding to the Hp-Hb complex eluted ~100 s later in ~14% of all study samples, which was not correlated with age or disease diagnosis, but is consistent with the presence of the smaller Hp (1-1) isoform in 15% of the population.

Toxic Metal Species and 'Endogenous' Metalloproteins at the Blood-Organ Interface: Analytical and Bioinorganic Aspects.

Globally, human exposure to environmental pollutants causes an estimated 9 million deaths per year and it could also be implicated in the etiology of diseases that do not appear to have a genetic origin. Accordingly, there is a need to gain information about the biomolecular mechanisms that causally link exposure to inorganic environmental pollutants with distinct adverse health effects. Although the analysis of blood plasma and red blood cell (RBC) cytosol can provide important biochemical information about these mechanisms, the inherent complexity of these biological matrices can make this a difficult task. In this perspective, we will examine the use of metalloentities that are present in plasma and RBC cytosol as potential exposure biomarkers to assess human exposure to inorganic pollutants. Our primary objective is to explore the principal bioinorganic processes that contribute to increased or decreased metalloprotein concentrations in plasma and/or RBC cytosol. Furthermore, we will also identify metabolites which can form in the bloodstream and contain essential as well as toxic metals for use as exposure biomarkers. While the latter metal species represent useful biomarkers for short-term exposure, endogenous plasma metalloproteins represent indicators to assess the long-term exposure of an individual to inorganic pollutants. Based on these considerations, the quantification of metalloentities in blood plasma and/or RBC cytosol is identified as a feasible research avenue to better understand the adverse health effects that are associated with chronic exposure of various human populations to inorganic pollutants. Exposure to these pollutants will likely increase as a consequence of technological advances, including the fast-growing applications of metal-based engineering nanomaterials.

Linking molecular targets of Cd in the bloodstream to organ-based adverse health effects.

The chronic exposure of human populations to toxic metals remains a global public health concern. Although chronic Cd exposure is linked to kidney damage, osteoporosis and cancer, the underlying biomolecular mechanisms remain incompletely understood. Since other diseases could also be causally linked to chronic Cd exposure, a systems toxicology-based approach is needed to gain new insight into the underlying exposure-disease relationship. This approach requires one to integrate the cascade of dynamic bioinorganic chemistry events that unfold in the bloodstream after Cd enters with toxicological events that unfold in target organs over time. To this end, we have conducted a systematic literature search to identify all molecular targets of Cd in plasma and in red blood cells (RBCs). Based on this information it is impossible to describe the metabolism of Cd and the toxicological relevance of it binding to molecular targets in/on RBCs is elusive. Perhaps most importantly, the role that peptides, amino acids and inorganic ions, including HCO3-, Cl- and HSeO3- play in terms of mediating the translocation of Cd to target organs and its detoxification is poorly understood. Causally linking human exposure to this metal with diseases requires a much better integration of the bioinorganic chemistry of Cd that unfolds in the bloodstream with target organs. This from a public health point of view important goal will require collaborations between scientists from different disciplines to untangle the complex mechanisms which causally link Cd exposure to disease.

Application of a Novel Metallomics Tool to Probe the Fate of Metal-Based Anticancer Drugs in Blood Plasma: Potential, Challenges and Prospects.

Although metallodrugs are used to treat a variety of human disorders and exhibit a remarkable diversity of therapeutic properties, they constitute only a tiny minority of all medicinal drugs that are currently on the market. This undesirable situation must be partially attributed to our general lack of understanding the fate of metallodrugs in the extremely ligand-rich environment of the bloodstream. The challenge of gaining insight into these bioinorganic processes can be overcome by the application of 'metallomics tools', which involve the analysis of biological fluids (e.g., blood plasma) with a separation method in conjunction with multi-element specific detectors. To this end, we have developed a metallomics tool that is based on size-exclusion chromatography (SEC) hyphenated to an inductively coupled plasma atomic emission spectrometer (ICP-AES). After the successful application of SEC-ICPAES to analyze plasma for endogenous copper, iron and zinc-metalloproteins, it was subsequently applied to probe the metabolism of a variety of metal-based anticancer drugs in plasma. The versatility of this metallomics tool is exemplified by the fact that it has provided insight into the metabolism of individual Pt-based drugs, the modulation of the metabolism of cisplatin by sulfur-containing compounds, the metabolism of two metal-based drugs that contain different metals as well as a bimetallic anticancer drug, which contained two different metals. After adding pharmacologically relevant doses of metallodrugs to plasma, the temporal analysis of aliquots by SEC-ICP-AES allows to observe metal-protein adducts, metallodrug-derived degradation products and the parent metallodrug(s). This unique capability allows to obtain comprehensive insight into the fate of metal-based drugs in plasma and can be extended to in vivo studies. Thus, the application of this metallomics tool to probe the fate of novel metalcomplexes that exert the desired biological activity in plasma has the potential to advance more of these to animal/preclinical studies to fully explore the potential that metallodrugs inherently offer.

Sample preparation of blood plasma enables baseline separation of iron metalloproteins by SEC-GFAAS.

The analysis of human plasma for biomarkers holds promise to revolutionize disease diagnosis, but is hampered by the inherent complexity of the plasma proteome. One way to overcome this problem is to analyze plasma for a sub-proteome, such as the metalloproteome. Previous studies employing size-exclusion chromatography (SEC) coupled on-line to an inductively coupled plasma-atomic emission spectrometer (ICP-AES) have revealed that plasma contains ~12 copper, iron and zinc metalloproteins. This included the iron metalloproteins transferrin (Tf) and a recently identified haptoglobin-hemoglobin (Hp-Hb) complex, which is formed in plasma when red blood cells rupture. Since this SEC-ICP-AES method required a sample volume of 500 µL to generate diagnostically useful results, we sought to develop an alternative SEC-based hyphenated approach using a smaller SEC column (150 × 5 mm I.D.) and a graphite furnace atomic absorption spectrometer (GFAAS) as the iron-specific detector. A designed interface enabled the integration of the SEC system with the GFAAS. Baseline separation between the Hp-Hb complex and Tf was achieved by developing a sample preparation procedure which involved the chelating agent-based mobilization of Fe from Tf to a small molecular weight Fe complex. Spiking of human plasma (1.0 mL) with red blood cell lysate (1-2 µL) increased only the intensity of the Fe peak corresponding to the Hp-Hb complex, but not that of Tf. Since the developed SEC-GFAAS method requires only 50 µL of plasma for analysis, it can now be employed for the cost-effective quantification of the clinically relevant Hb-Hp complex in human plasma in <50 min.

SEC hyphenated to a multielement-specific detector unravels the degradation pathway of a bimetallic anticancer complex in human plasma.

The bimetallic metal complex Titanocref exhibits relevant anticancer activity, but it is unknown if it is stable to reach target tissues intact. To gain insight, a pharmacologically relevant dose was added to human blood plasma and the mixture was incubated at 37 °C. The obtained mixture was analyzed 5 and 60 min later by size-exclusion chromatography hyphenated to an inductively coupled plasma atomic emission spectrometer (SEC-ICP-AES). We simultaneously detected several titanium (Ti), gold (Au) and sulfur (S)-peaks, which corresponded to a Ti degradation product that eluted partially, and a Au degradation product that eluted entirely bound to plasma proteins (both time points). Although ~70% of the intact Titanocref was retained on the column after 60 min, our results allowed us to establish - for the first time - its likely degradation pathway in human plasma at near physiological conditions. These results suggest that ~70% of Titanocref remain in plasma after 60 min, which supports results from a recent in vivo study in which mice were treated with Titanocref and revealed Ti:Au molar ratios in tumors and organs close to 1:1. Thus, our stability studies suggest that the intact drug is able to reach target tissue. Overall, our results exemplify that SEC-ICP-AES enables the execution of intermediate in vitro studies with human plasma in the context of advancing bimetallic metal-based drugs to more costly clinical studies.

Identification of a haptoglobin-hemoglobin complex in human blood plasma.

Blood plasma metalloproteins that contain copper (Cu), iron (Fe), zinc (Zn) and/or other metals/metalloids are potential disease biomarkers because the bloodstream is in permanent contact with organs. Their quantification and/or the presence of additional metal-entities or the absence of certain metalloproteins in blood plasma (e.g. in Wilson's disease) may provide insight into the dyshomeostasis of the corresponding metal (s) to gain insight into disease processes. The first step in investigating if the determination of plasma metalloproteins is useful for the diagnosis of diseases is their definitive qualitative identification. To this end, we have added individual highly pure Cu, Fe or Zn-containing metalloproteins to plasma (healthy volunteer) and analyzed this mixture by size-exclusion chromatography (SEC) coupled to an inductively coupled plasma atomic spectrometer (ICP-AES), simultaneously monitoring the emission lines of Cu, Fe and Zn. The results clearly identified ceruloplasmin (Cp), holo-transferrin (hTf), and α2-macroglobulin (α2M), which verifies our previous assignments. Interestingly, another major Fe-peak in plasma was identified as a haptoglobin (Hp)-hemoglobin (Hb) complex. This Hp-Hb complex is formed after Hb, which is released during the hemolysis of erythrocytes, binds to the plasma protein Hp. The Hp-Hb complex formation is known to be one of the strongest interactions in biochemistry (Kd≈1pmol/L) and is critical because it prevents kidney toxicity of free Hb. Hence, the simultaneous determination of Cp, hTf, α2M and the Hp-Hb complex in plasma in <25min has the potential to provide new insight into disease processes associated with the bioinorganic chemistry of Cu, Fe and Zn.

Improving the safety of metal-based drugs by tuning their metabolism with chemoprotective agents.

Metal-based drugs remain a tiny minority of all drugs that are on the market. The success story of the quintessential metal-based drug cisplatin (CP), which is intravenously administered to 70% of all cancer patients, however, demonstrates the inherent potential of metal-based drugs. A distinct disadvantage of CP is the dose-limiting severe toxic-side effects that it exerts in patients. To better understand the biomolecular basis for its toxicity, we employed a metallomics method to observe all platinum metabolites that are formed in blood plasma. These investigations revealed that a highly toxic CP-derived hydrolysis product - the highly toxic monoaqua hydrolysis complex (MHC) - is formed within 5min. More importantly, the application of this research tool has unraveled the mechanisms by which the chemoprotective agents sodium thiosulfate, d-methionine, N-acetyl-cysteine and l-glutathione modulate the metabolism of CP in plasma, namely by rapidly reacting with the MHC to form platinum­sulfur complexes. Since CP remained in plasma for a considerable time, the possibility of 'tuning' its metabolism with chemoprotective agents in a desirable way has emerged. These observations are highly relevant because these chemoprotective agents were previously shown to significantly reduce the toxicity of CP in animal models, often without appreciably affecting its anticancer efficiency. Collectively, these results suggest that the toxicity of other metal-based drugs may be overcome if their metabolism in the bloodstream is adequately tuned with a suitable chemoprotective agent. This principle strategy has considerable potential in terms of harnessing the full potential of bringing more metal-based drugs to the market.

Selenium-mediated arsenic excretion in mammals: a synchrotron-based study of whole-body distribution and tissue-specific chemistry.

Arsenicosis, a syndrome caused by ingestion of arsenic contaminated drinking water, currently affects millions of people in South-East Asia and elsewhere. Previous animal studies revealed that the toxicity of arsenite essentially can be abolished if selenium is co-administered as selenite. Although subsequent studies have provided some insight into the biomolecular basis of this striking antagonism, many details of the biochemical pathways that ultimately result in the detoxification and excretion of arsenic using selenium supplements have yet to be thoroughly studied. To this end and in conjunction with the recent Phase III clinical trial "Selenium in the Treatment of Arsenic Toxicity and Cancers", we have applied synchrotron X-ray techniques to elucidate the mechanisms of this arsenic-selenium antagonism at the tissue and organ levels using an animal model. X-ray fluorescence imaging (XFI) of cryo-dried whole-body sections of laboratory hamsters that had been injected with arsenite, selenite, or both chemical species, provided insight into the distribution of both metalloids 30 minutes after treatment. Co-treated animals showed strong co-localization of arsenic and selenium in the liver, gall bladder and small intestine. X-ray absorption spectroscopy (XAS) of freshly frozen organs of co-treated animals revealed the presence in liver tissues of the seleno bis-(S-glutathionyl) arsinium ion, which was rapidly excreted via bile into the intestinal tract. These results firmly support the previously postulated hepatobiliary excretion of the seleno bis-(S-glutathionyl) arsinium ion by providing the first data pertaining to organs of whole animals.

Remarkable differences in the biochemical fate of Cd2+, Hg2+, CH3Hg+ and thimerosal in red blood cell lysate.

Humans are environmentally exposed to potentially toxic Cd and Hg species and to the Hg compound thimerosal (THI), an antibactericidal vaccine additive. Previous studies have revealed that Cd2+, Hg2+ and CH3Hg+ are taken up by red blood cells (RBCs) and bind to cytosolic glutathione (GSH) and/or hemoglobin (Hb). Since interactions in the cytosol of RBCs may be linked to their hemolysis, a more comprehensive characterization of these interactions was sought. After the addition of each Cd and Hg species to RBC lysate, the mixtures were analyzed after 5 min, 2 h and 6 h by size-exclusion chromatography (SEC) coupled on-line to an inductively coupled plasma atomic emission spectrometer (ICP-AES). In contrast to previous studies, however, reducing conditions were maintained by employing a 100 mM Tris buffer mobile phase (pH 7.4), which contained ∼2.5 mM of glutathione (GSH). At ≥2 h, ∼85% of Cd2+ weakly interacted with hemoglobin (Hb), while ∼13% eluted as (GS)xCd and ∼2% bound to a ≥70 kDa Cd-binding protein. In contrast, ∼6% of Hg2+ co-eluted with Hb at all time points, while ∼94% eluted as (GS)xHg. The results for CH3Hg+ showed that ∼5% of Hg co-eluted with Hb, while for THI this percentage gradually increased to 12% (6 h). The remaining Hg eluted as GS-HgCH3 and GS-HgCH2CH3. Our results revealed remarkable differences in the interaction of the investigated Cd and Hg species with cytosolic RBC constituents. The formation of (Hb)xHg species, regardless of which Hg compound was added, suggests their mammalian toxicology to be intertwined with the metabolism of Fe.

Environmentally relevant concentrations of aminopolycarboxylate chelating agents mobilize Cd from humic acid.

Although Cd is a pollutant of public health relevance, many dietary sources from which it can be absorbed into human tissues remain unknown. While it is well established that the biogeochemical cycle of Cd involves its complexation with environment-derived ligands (e.g., humic acids, HAs) and anthropogenic ones (e.g., chelating agents, CAs), the interaction of Cd with both of these ligands is less well understood. To gain insight, a HA-Cd complex was injected on a size-exclusion chromatography (SEC) column coupled on-line with a flame atomic absorption spectrometer (FAAS) using 10mmol/L Tris buffer (pH8.0) as the mobile phase. This approach allowed us to observe the intact HA-Cd complex and the retention behavior of Cd as a function of 2-20µmol/L concentrations of ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA) or methylglycinediacetic acid (MGDA) that were added to the mobile phase. An increase of the retention time of Cd was indicative of a partial or complete abstraction of Cd from HA. Our results revealed that all CAs abstracted Cd from the HA-Cd complex at concentrations of 5µmol/L, while MGDA and DTPA were effective at 2µmol/L. The bioavailability of some of the on-column formed CA-Cd complexes explains the previously reported increased accumulation of Cd in periphyton in the ecosystem downstream of wastewater treatment plants. In addition, our results imply that the use of effluents which contain CAs and Cd for the irrigation of food crops can introduce Cd into the food supply and compromise food safety.

Tuning the metabolism of the anticancer drug cisplatin with chemoprotective agents to improve its safety and efficacy.

Numerous in vivo studies have shown that the severe toxic side-effects of intravenously administered cisplatin can be significantly reduced by the co-administration of sulfur-containing 'chemoprotective agents'. Using a metallomics approach, a likely biochemical basis for these potentially useful observations was only recently uncovered and appears to involve the reaction of chemoprotective agents with cisplatin-derived Pt-species in human plasma to form novel platinum-sulfur complexes (PSC's). We here reveal aspects of the structure of two PSC's and establish the identification of an optimal chemoprotective agent to ameliorate the toxic side-effects of cisplatin, while leaving its antineoplastic activity largely intact, as a feasible research strategy to transform cisplatin into a safer and more effective anticancer drug.