Extracellular Calcium Receptor as a Target for Glutathione and Its Derivatives

Extracellular glutathione (GSH) and oxidized glutathione (GSSG) can modulate the function of the extracellular calcium sensing receptor (CaSR). The CaSR has a binding pocket in the extracellular domain of CaSR large enough to bind either GSH or GSSG, as well as the naturally occurring oxidized derivative L-cysteine glutathione disulfide (CySSG) and the compound cysteinyl glutathione (CysGSH). Modeling the binding energies (ΔG) of CySSG and CysGSH to CaSR reveals that both cysteine derivatives may have greater affinities for CaSR than either GSH or GSSG. GSH, CySSG, and GSSG are found in circulation in mammals and, among the three, CySSG is more affected by HIV/AIDs and aging than either GSH or GSSG. The beta-carbon linkage of cysteine in CysGSH may model a new class of calcimimetics, exemplified by etelcalcetide. Circulating glutathionergic compounds, particularly CySSG, may mediate calcium-regulatory responses via receptor-binding to CaSR in a variety of organs, including parathyroids, kidneys, and bones. Receptor-mediated actions of glutathionergics may thus complement their roles in redox regulation and detoxification. The glutathionergic binding site(s) on CaSR are suggested to be a target for development of drugs that can be used in treating kidney and other diseases whose mechanisms involve CaSR dysregulation.

Bioremediation of arsenic contamination from the environment: New approach to sustainable resource management

Present acceleration of Arsenic [As] exposure leads to severe health problems. Modern scientific approaches look towards potent bio-agents for the removal of such types of contaminations in sustainable ways. Microbes can potentially change the redox potential, solubility, pH by different complex reactions during bioremediation. There are many enzymes present in the microbial system which are involved in methylation such as As (V) reductase, monomethyl arsonic acid reductase, As (III) methyltransferase, and MMA (III) methyltransferase. On the other hand, microbes have As transformation ability and changed into different extractable forms with sulfide minerals such as arsenopyrite (FeAsS), enargite (Cu3AsS4) and realgar (As4S4). In some bacteria, the As-operon machinery thiol group bind with As, itdetoxifies its toxicity. Ars R gene and arsenic reductase enzyme (Ars C) play the key role in the reduction of As (V) to As (III) and detoxify by being transported outside of the cell by Ars AB As chemiosmotic efflux system. In fungi, As (V) is reduced to As (III) by the arsenate reductase and GSH glutathione converted into GSSH glutathione disulfide. In plants, As (III) conjugates with phytochelatin (PC) or GSH glutathione and accumulates in the vacuole or is converted into less toxic forms in the presence of arsenic reductase enzyme. This review focused on the potentiality and mechanisms of different microbes for As-detoxification in a sustainable manner.

BIOM-13. DEVELOPMENT AND VALIDATION OF AN LC-MS/MS ASSAY TO MEASURE GLUTATHIONE, GLUTATHIONE DISULFIDE, CYSTEINE, AND CYSTINE IN HUMAN BRAIN AND HUMAN GLIOBLASTOMA

BACKGROUND Oxidative stress is implicated in many pathological conditions. Herein, we report on our development and validation of a sensitive and rapid LC-MS/MS method for the determination of oxidative stress biomarkers glutathione (GSH), glutathione disulfide (GSSG), cysteine (Cys) and cystine (CySS) in human brain and glioblastoma tissue. METHODS Freshly-acquired human glioblastoma tissue was homogenized with N-ethylmaleimide solution to prevent thiol oxidation. Analytes were then extracted from homogenate samples by protein precipitation with 2% sulfosalicylic acid (SSA). Stable isotope-labeled analytes were used as internal standards. Independent calibration curves for thiols and disulfides were prepared in analytical solutions. Three levels of quality controls were prepared in human brain homogenate. The detection was performed on Sciex QTRAP 6500+ mass spectrometer in positive electrospray ionization mode. RESULTS Linear regression model was used to cover a concentration ranging 0.4-100 µmol/L for GSSG/CySS and 1-400 µmol/L for GSH/Cys. Chromatographic separation was optimized on Intrada Amino Acid column with total run time of 5 min using gradient elution. For all analytes the maximum coefficient of variation for intra- and inter-day precision was 11.4% and the accuracy was within 80.9-113.7% in analytical solution and matrix. The analytes were stable in brain homogenate for 1 hour and 3 hours at room temperature (RT) and 4 °C, respectively. Stability at -80°C was demonstrated for at least 35 days in human brain homogenate. Stability of stock and working solutions was demonstrated for at least 4 hours (RT) and 25 days (-20°C). CONCLUSIONS A bioanalytical method to quantify GSH, GSSG, Cys, and CySS is successfully developed and validated. The method is currently applied to measure thiols and related disulfides in human glioblastoma tissue undergoing 5-aminolevulinic acid sonodynamic therapy (NCT04559685).

Silver Nanoparticle-Mediated Synthesis of Fluorescent Thiolated Gold Nanoclusters

A new strategy using silver nanoparticles (Ag NPs) to synthesize thiolated Au NCs is demonstrated. The quasi-spherical Ag NPs serve as a platform, functioning as a reducing agent for Au (III) and attracting capping ligands to the surface of the Ag NPs. Glutathione disulfide (GSSG) and dithiothreitol (DTT) were used as capping ligands to synthesize thiolated Au NCs (glutathione-Au NCs and DTT-Au NCs). The glutathione-Au NCs and DTT-Au NCs showed red color luminance with similar emission wavelengths (630 nm) at an excitation wavelength of 354 nm. The quantum yields of the glutathione-Au NCs and DTT-Au NCs were measured to be 7.3% and 7.0%, respectively. An electrophoretic mobility assay showed that the glutathione-Au NCs moved toward the anode, while the DTT-Au NCs were not mobile under the electric field, suggesting that the total net charge of the thiolated Au NCs is determined by the charges on the capping ligands. The detection of the KSV values, 26 M−1 and 0 M−1, respectively, revealed that glutathione-Au NCs are much more accessible to an aqueous environment than DTT-Au NCs.

Glutathione Disulfide as a Reducing, Capping, and Mass-Separating Agent for the Synthesis and Enrichment of Gold Nanoclusters

Water-soluble nanoclusters, which are facilely enrichable without changes in the original properties, are highly demanded in many disciplines. In this contribution, a new class of gold nanoclusters (AuNCs) was synthesized using glutathione disulfide (GSSG) as a reducing and capping agent under intermittent heating mode. The as-prepared GSSG–AuNCs had a higher quantum yield (4.1%) compared to the conventional glutathione-protected AuNCs (1.8%). Moreover, by simply introducing the GSSG–AuNC solution to acetonitrile at a volume ratio of 1:7, a new bottom phase was formed, in which GSSG–AuNCs could be 400-fold enriched without changes in properties, with a percentage recovery higher than 99%. The enrichment approach did not need additional instruments and was potentially suitable for large-scale enrichment of nanoclusters. Further, density functional theory calculations indicated that the hydrogen bonding between GSSG and acetonitrile plays a key role for the bottom phase formation. Our work suggests that the highly emissive GSSG–AuNCs possess great potential not only in fluorescent measurements but also in other scenarios in which high-concentration AuNCs may be needed, such as catalysis, drug delivery, and electronic and optical industries.

The Redox Potential of the β-93-Cysteine Thiol Group in Human Hemoglobin Estimated from In Vitro Oxidant Challenge Experiments

Glutathionyl hemoglobin is a minor form of hemoglobin with intriguing properties. The measurement of the redox potential of its reactive β-93-Cysteine is useful to improve understanding of the response of erythrocytes to transient and chronic conditions of oxidative stress, where the level of glutathionyl hemoglobin is increased. An independent literature experiment describes the recovery of human erythrocytes exposed to an oxidant burst by measuring glutathione, glutathione disulfide and glutathionyl hemoglobin in a two-hour period. This article calculates a value for the redox potential E0 of the β-93-Cysteine, considering the erythrocyte as a closed system at equilibrium described by the Nernst equation and using the measurements of the literature experiment. The obtained value of E0 of −121 mV at pH 7.4 places hemoglobin as the most oxidizing thiol of the erythrocyte. By using as synthetic indicators of the concentrations the electrochemical potentials of the two main redox pairs in the erythrocytes, those of glutathione–glutathione disulfide and of glutathionyl–hemoglobin, the mechanism of the recovery phase can be hypothesized. Hemoglobin acts as the redox buffer that scavenges oxidized glutathione in the oxidative phase and releases it in the recovery phase, by acting as the substrate of the NAD(P)H-cofactored enzymes.

Simultaneous Biochemical and Physiological Responses of the Roots and Leaves of Pancratium maritimum (Amaryllidaceae) to Mild Salt Stress

Pancratium maritimum (Amaryllidaceae) is a bulbous geophyte growing on coastal sands. In this study, we investigated changes in concentrations of metabolites in the root and leaf tissue of P. maritimum in response to mild salt stress. Changes in concentrations of osmolytes, glutathione, sodium, mineral nutrients, enzymes, and other compounds in the leaves and roots were measured at 0, 3, and 10 days during a 10-day exposure to two levels of mild salt stress, 50 mM NaCl or 100 mM NaCl in sandy soil from where the plants were collected in dunes near Cuma, Italy. Sodium accumulated in the roots, and relatively little was translocated to the leaves. At both concentrations of NaCl, higher values of the concentrations of oxidized glutathione disulfide (GSSG), compared to reduced glutathione (GSH), in roots and leaves were associated with salt tolerance. The concentration of proline increased more in the leaves than in the roots, and glycine betaine increased in both roots and leaves. Differences in the accumulation of organic osmolytes and electron donors synthesized in both leaves and roots demonstrate that osmoregulatory and electrical responses occur in these organs of P. maritimum under mild salt stress.

Glutathione disulfide-sensitive Janus nanomachine controlled by an enzymatic AND logic gate for smart delivery

This work describes the assembly of a novel enzyme-controlled nanomachine operated through an AND Boolean logic gate for on-command delivery. The nanodevice was constructed on Au-mesoporous silica Janus nanoparticles capped...