Insights into the trypanothione system in antimony-resistant and sensitive Leishmania tropica clinical isolates.

Pentavalent antimonials are the mainstay treatment against different clinical forms of leishmaniasis. The emergence of resistant isolates in endemic areas has led to treatment failure. Unraveling the underlying resistance mechanism would assist in improving the treatment strategies against resistant isolates. This study aimed to investigate the RNA expression level of glutathione synthetase (GS), Spermidine synthetase (SpS), trypanothione synthetase (TryS) genes involved in trypanothione synthesis, and thiol-dependent reductase (TDR) implicated in drug reduction, in antimony-sensitive and -resistant Leishmania tropica isolates. We investigated 11 antimony-resistant and 11 antimony-sensitive L. tropica clinical isolates from ACL patients. Drug sensitivity of amastigotes was determined in mouse macrophage cell line J774A.1. The RNA expression level in the promastigote forms was analyzed by quantitative real-time PCR. The results revealed a significant increase in the average expression of GS, SpS, and TrpS genes by 2.19, 1.56, and 2.33-fold in resistant isolates compared to sensitive ones. The average expression of TDR was 1.24-fold higher in resistant isolates, which was insignificant. The highest correlation coefficient between inhibitory concentration (IC50) values and gene expression belonged to the TryS, GS, SpS, and TDR genes. Moreover, the intracellular thiol content was increased 2.17-fold in resistant isolates compared to sensitive ones and positively correlated with IC50 values. Our findings suggest that overexpression of trypanothione biosynthesis genes and increased thiol content might play a key role in the antimony resistance of L. tropica clinical isolates. In addition, the diversity of gene expression in the trypanothione system and thiol content among L. tropica clinical isolates highlighted the phenotypic heterogeneity of antimony resistance among the parasite population.

Emerging Glycation-Based Therapeutics-Glyoxalase 1 Inducers and Glyoxalase 1 Inhibitors.

The abnormal accumulation of methylglyoxal (MG) leading to increased glycation of protein and DNA has emerged as an important metabolic stress, dicarbonyl stress, linked to aging, and disease. Increased MG glycation produces inactivation and misfolding of proteins, cell dysfunction, activation of the unfolded protein response, and related low-grade inflammation. Glycation of DNA and the spliceosome contribute to an antiproliferative and apoptotic response of high, cytotoxic levels of MG. Glyoxalase 1 (Glo1) of the glyoxalase system has a major role in the metabolism of MG. Small molecule inducers of Glo1, Glo1 inducers, have been developed to alleviate dicarbonyl stress as a prospective treatment for the prevention and early-stage reversal of type 2 diabetes and prevention of vascular complications of diabetes. The first clinical trial with the Glo1 inducer, trans-resveratrol and hesperetin combination (tRES-HESP)-a randomized, double-blind, placebo-controlled crossover phase 2A study for correction of insulin resistance in overweight and obese subjects, was completed successfully. tRES-HESP corrected insulin resistance, improved dysglycemia, and low-grade inflammation. Cell permeable Glo1 inhibitor prodrugs have been developed to induce severe dicarbonyl stress as a prospective treatment for cancer-particularly for high Glo1 expressing-related multidrug-resistant tumors. The prototype Glo1 inhibitor is prodrug S-p-bromobenzylglutathione cyclopentyl diester (BBGD). It has antitumor activity in vitro and in tumor-bearing mice in vivo. In the National Cancer Institute human tumor cell line screen, BBGD was most active against the glioblastoma SNB-19 cell line. Recently, potent antitumor activity was found in glioblastoma multiforme tumor-bearing mice. High Glo1 expression is a negative survival factor in chemotherapy of breast cancer where adjunct therapy with a Glo1 inhibitor may improve treatment outcomes. BBGD has not yet been evaluated clinically. Glycation by MG now appears to be a pathogenic process that may be pharmacologically manipulated for therapeutic outcomes of potentially important clinical impact.

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.

Quantification of intracellular HNO delivery with capillary zone electrophoresis.

Redox signaling, wherein reactive and diffusible small molecules are channeled into specific messenger functions, is a critical component of signal transduction. A central principle of redox signaling is that the redox modulators are produced in a highly controlled fashion to specifically modify biotargets. Thiols serve as primary mediators of redox signaling as a function of the rich variety of adducts, which allows initiation of distinct cellular effects. Coupling the inherent reactivity of thiols with highly sensitive and selective chemical analysis protocols can facilitate identification of redox signaling agents, both in solution and in cultured cells. Here, we describe use of capillary zone electrophoresis to both identify and quantify sulfinamides, which are specific markers of the reaction of thiols with nitroxyl (HNO), a putative biologically relevant reactive nitrogen species.

Glutathione Trisulfide Prevents Lipopolysaccharide-induced Inflammatory Gene Expression in Retinal Pigment Epithelial Cells.

We investigated the effects of glutathione trisulfide (GSSSG) on lipopolysaccharide (LPS)-induced inflammatory gene expression in immortalized ARPE-19, and primary human and mouse retinal pigment epithelial (RPE) cells. Sulfane sulfur molecules were significantly increased in GSSSG-treated ARPE-19 cells. GSSSG prevented the LPS-induced upregulation of interleukin (IL)-1ß, IL-6, and C-C motif chemokine ligand 2 (CCL2) in ARPE-19/primary RPE cells. Moreover, GSSSG prevented the activation of the nuclear factor-kappa B p65 subunit, and promoted the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in LPS-treated ARPE-19 cells. ERK1/2 inhibition prevented the GSSSG-mediated inhibition of LPS-induced IL-6 and CCL2 upregulation. Additionally, ERK1/2 activation prevented the upregulation of these genes in the absence of GSSSG. Knockdown of HMOX1 or NRF2, known as anti-oxidative genes, did not affect the activity of GSSSG in the context of LPS stimulation. These findings suggest that GSSSG attenuates LPS-induced inflammatory gene expression via ERK signaling hyperactivation, independently of the NRF2/HMOX1 pathway.

Antitumor Effects of Selenium.

Functions of selenium are diverse as antioxidant, anti-inflammation, increased immunity, reduced cancer incidence, blocking tumor invasion and metastasis, and further clinical application as treatment with radiation and chemotherapy. These functions of selenium are mostly related to oxidation and reduction mechanisms of selenium metabolites. Hydrogen selenide from selenite, and methylselenol (MSeH) from Se-methylselenocyteine (MSeC) and methylseleninicacid (MSeA) are the most reactive metabolites produced reactive oxygen species (ROS); furthermore, these metabolites may involve in oxidizing sulfhydryl groups, including glutathione. Selenite also reacted with glutathione and produces hydrogen selenide via selenodiglutathione (SeDG), which induces cytotoxicity as cell apoptosis, ROS production, DNA damage, and adenosine-methionine methylation in the cellular nucleus. However, a more pronounced effect was shown in the subsequent treatment of sodium selenite with chemotherapy and radiation therapy. High doses of sodium selenite were effective to increase radiation therapy and chemotherapy, and further to reduce radiation side effects and drug resistance. In our study, advanced cancer patients can tolerate until 5000 µg of sodium selenite in combination with radiation and chemotherapy since the half-life of sodium selenite may be relatively short, and, further, selenium may accumulates more in cancer cells than that of normal cells, which may be toxic to the cancer cells. Further clinical studies of high amount sodium selenite are required to treat advanced cancer patients.

Characterization of primary glutathione conjugates with acrylamide and glycidamide: Toxicokinetic studies in Sprague Dawley rats treated with acrylamide.

Acrylamide (AA) is classified as a probable human carcinogen and is ubiquitous in foods processed at high temperatures. The carcinogenicity of AA has been attributed to its active metabolite, glycidamide (GA). Both AA and GA can spontaneously or enzymatically conjugate with glutathione (GSH) to form their corresponding GSH conjugates. Profiling AA-glutathione conjugate (AA-GSH) and GA-glutathione conjugates (2 isomers: GA2-GSH and GA3-GSH) in serum would better illustrate AA detoxification compared with urinary metabolite analysis. However, the lack of AA-, GA2, and GA3-GSH study remains a critical data gap. Our study aimed to investigate the toxicokinetics of AA-, GA2-and GA3-GSH in Sprague Dawley rats treated with 0.1 mg/kg, 1.0 mg/kg, or 5.0 mg/kg AA. Blood samples were collected for LC-MS/MS analysis of the GSH conjugate products. Within 24 h of treatment, we observed rapid formation, elimination, and linear kinetics of AA-, GA2-and GA3-GSH. The ∑GA-GSH AUC/AA-GSH AUC ratios were 0.14-0.29, similar to ∑GA/AA AUC in serum but different from ∑GA/AA-derived urinary mercapturic acids in rodents. Our analysis of AA- and GA-GSHs values represents direct detoxification of AA and GA in vivo. This study advances our understanding of sex and inter-species differences in AA detoxification and may refine the existing kinetic models for a more relevant risk extrapolation.

Dual Activity BLEG-1 from Bacillus lehensis G1 Revealed Structural Resemblance to B3 Metallo-ß-Lactamase and Glyoxalase II: An Insight into Its Enzyme Promiscuity and Evolutionary Divergence.

Metallo-ß-lactamases (MBLs) are class B ß-lactamases from the metallo-hydrolase-like MBL-fold superfamily which act on a broad range of ß-lactam antibiotics. A previous study on BLEG-1 (formerly called Bleg1_2437), a hypothetical protein from Bacillus lehensis G1, revealed sequence similarity and activity to B3 subclass MBLs, despite its evolutionary divergence from these enzymes. Its relatedness to glyoxalase II (GLXII) raises the possibility of its enzymatic promiscuity and unique structural features compared to other MBLs and GLXIIs. This present study highlights that BLEG-1 possessed both MBL and GLXII activities with similar catalytic efficiencies. Its crystal structure revealed highly similar active site configuration to YcbL and GloB GLXIIs from Salmonella enterica, and L1 B3 MBL from Stenotrophomonas maltophilia. However, different from GLXIIs, BLEG-1 has an insertion of an active-site loop, forming a binding cavity similar to B3 MBL at the N-terminal region. We propose that BLEG-1 could possibly have evolved from GLXII and adopted MBL activity through this insertion.

Metabolic Activation of Atomoxetine Mediated by Cytochrome P450 2D6.

Atomoxetine (ATX) is a neurological drug widely used for the treatment of attention deficit-hyperactivity disorder. Liver injury has been documented in patients administered ATX. The mechanism of ATX's toxic action is less clear. This study is aimed to characterize reactive metabolites of ATX in vitro and in vivo to assist our understanding of the mechanisms of ATX hepatotoxicity. A hydroxylated metabolite, along with an O-dealkylation metabolite, was found in ATX-supplemented rat liver microsome incubations. Additionally, two glutathione (GSH) conjugates and two N-acetylcysteine (NAC) conjugates were observed in rat liver microsome incubations containing ATX, NADPH, and GSH or NAC. The corresponding GSH conjugates and NAC conjugates were found in bile and urine of ATX-treated rats, respectively. Recombinant P450 enzyme incubation study demonstrated that CYP2D6 dominated the metabolic activation of ATX. The insights gained from this study may be of assistance to illuminate the mechanisms of ATX-induced hepatotoxicity.

[Roles of Gut Microflora in Selenium Metabolism of Host Animals].

Selenium (Se) shows biologically ambivalent characteristics in animals. It is an essential element but becomes severely toxic when the amount ingested exceeds the adequate intake level. Animals must be able to metabolize the various selenocompounds in meat, fish and vegetables to utilize Se for selenoprotein synthesis. It is known that the biological, nutritional, and toxicological effects of Se are strongly dependent on its chemical form. First, we evaluated the nutritional availability of nine naturally occurring Se compounds, or the so-called bioselenocompounds, in vivo. Second, we evaluated that gut microflora might contributes to the Se nutritional availability. Se is mainly excreted into urine. However, a substantial amount of Se was secreted into bile although Se was hardly detected in feces. Third, we evaluated the biological significance of biliary secretion of Se in terms of mineral nutrition. Finally, we discussed the entire Se metabolism in gut contributing to Se homeostasis in animal.

The thiol-based reduction of Bi(V) and Sb(V) anti-leishmanial complexes.

Low molecular weight thiols including trypanothione and glutathione play an important function in the cellular growth, maintenance and reduction of oxidative stress in Leishmania species. In particular, parasite specific trypanothione has been established as a prime target for new anti-leishmania drugs. Previous studies into the interaction of the front-line Sb(V) based anti-leishmanial drug meglumine antimoniate with glutathione, have demonstrated that a reduction pathway may be responsible for its effective and selective nature. The new suite of organometallic complexes, of general formula [MAr3(O2CR)2] (M = Sb or Bi) have been shown to have potential as new selective drug candidates. However, their behaviour towards the critical thiols glutathione and trypanothione is still largely unknown. Using NMR spectroscopy and mass spectrometry we have examined the interaction of the analogous Sb(V) and Bi(V) organometallic complexes, [SbPh3(O2CCH2(C6H4CH3))2] S1 and [BiPh3(O2CCH2(C6H4CH3))2] B1, with the trifluoroacetate (TFA) salt of trypanothione and L-glutathione. In the presence of trypanothione or glutathione at the clinically relevant pH of 4-5 for Leishmania amastigotes, both complexes undergo facile and rapid reduction, with no discernible difference. However, at a higher pH (6-7), the complexes behave quite differently towards glutathione. The Bi(V) complex is again reduced rapidly but the Sb(V) complex undergoes slow reduction over 8 h (t1/2 = 54 min.) These results give the first insights into why the highly oxidising Bi(V) complexes display low selectivity in their cytotoxicity towards leishmanial and mammalian cells, while the Sb(V) complexes show good selectivity.

The effects of glutathione ethyl ester in in vitro maturation on the developmental ability of oocytes derived from cattle with liver abnormalities.

The main objectives of this study was to identify the effects of a relationship of hyper-concentration of Gamma-glutamyltransferase (γ-GTP) in follicle fluid (FF) on the levels of glutathione (GSH)/reactive oxygen species (ROS) in oocytes and subsequent embryo development in cattle with abnormal livers. Furthermore, we investigated the effect of supplementing in vitro maturation medium with glutathione ethyl ester (GSH-OEt) on the subsequent developmental potential of oocytes from such cattle. We used a control group of cattle (with normal livers) and a liver disorder (LD) group, in which the liver was diagnosed as being abnormal. In experiment 1, the LD group was divided to two subgroups according to the concentration of γ-GTP in FF: a low group (≤50 IU/L; the low LD group), and a high group (>50 IU/L: the high LD group). Cumulus oocyte-complexes (COCs) were matured and fertilized in vitro and then cultured to the blastocyst stage. The levels of GSH and ROS in the matured oocytes after IVM were then assessed in each group. On day 7 after fertilization, embryo cleavage and development were assessed. We found that the rate of development to the blastocyst stage was significantly lower in the high LD group than in the control group and the low LD group. The levels of GSH in matured oocytes were significantly lower in the high LD group than in the control group and low LD group. The levels of ROS in matured oocytes was significantly higher in the high LD group than in the control group and the low LD group. In experiment 2, COCs from cattle in the high LD group were matured in m-199 supplemented with 5 mM GSH-OEt, then IVF and IVC was performed for 7 days. The GSH levels were determined in some COCs after IVM. The supplementation of media with GSH-OEt during IVM increased the levels of GSH in mature oocytes and improved the rate of blastocyst development compared with the control group. In conclusion, GSH-OEt supplementation to media during IVM can improve the developmental potential of oocytes in liver-diseased cattle with high γ-GTP concentrations in the FF by increasing intracellular GSH synthesis and scavenging ROS.

Glutathione ethyl ester reverses the deleterious effects of fentanyl on ventilation and arterial blood-gas chemistry while prolonging fentanyl-induced analgesia.

There is an urgent need to develop novel compounds that prevent the deleterious effects of opioids such as fentanyl on minute ventilation while, if possible, preserving the analgesic actions of the opioids. We report that L-glutathione ethyl ester (GSHee) may be such a novel compound. In this study, we measured tail flick latency (TFL), arterial blood gas (ABG) chemistry, Alveolar-arterial gradient, and ventilatory parameters by whole body plethysmography to determine the responses elicited by bolus injections of fentanyl (75 µg/kg, IV) in male adult Sprague-Dawley rats that had received a bolus injection of GSHee (100 µmol/kg, IV) 15 min previously. GSHee given alone had minimal effects on TFL, ABG chemistry and A-a gradient whereas it elicited changes in some ventilatory parameters such as an increase in breathing frequency. In vehicle-treated rats, fentanyl elicited (1) an increase in TFL, (2) decreases in pH, pO2 and sO2 and increases in pCO2 (all indicative of ventilatory depression), (3) an increase in Alveolar-arterial gradient (indicative of a mismatch in ventilation-perfusion in the lungs), and (4) changes in ventilatory parameters such as a reduction in tidal volume, that were indicative of pronounced ventilatory depression. In GSHee-pretreated rats, fentanyl elicited a more prolonged analgesia, relatively minor changes in ABG chemistry and Alveolar-arterial gradient, and a substantially milder depression of ventilation. GSHee may represent an effective member of a novel class of thiolester drugs that are able to prevent the ventilatory depressant effects elicited by powerful opioids such as fentanyl and their deleterious effects on gas-exchange in the lungs without compromising opioid analgesia.

Glutathione ethyl ester improved the age-induced decline in the developmental competence of bovine oocytes.

The aberrant redox regulation and anti-oxidative defense is one of the main causes of age-induced decline in oocytes quality and embryo development in mammals. The present study aimed to elucidate the effect of glutathione ethyl ester (GSH-OEt), a cell-permeable glutathione (GSH) donor, on the developmental competence of oocytes in cows with advanced reproductive age. Oocytes were collected from cows aged 30-50 months or >120 months, which were defined as young or aged, respectively, and subjected to in vitro maturation (IVM) in the presence of 5 mM of GSH-OEt. In aged cows, the GSH level in follicular fluid was lower, and the intracellular levels of reactive oxygen species (ROS) in post-IVM oocytes was higher than those in young cows. GSH-OEt supplementation during IVM reduced the ROS contents of oocyte in aged cows but not in young cows. GSH-OEt treatment promoted the meiotic progression and increased the proportion of oocytes with mature cytoplasm containing evenly dispersed cortical granules in aged cows. After in vitro fertilization, the normal fertilization and development to the blastocyst stage were enhanced by GSH-OEt in aged cows to levels comparable to those in young cows. Further, oocyte maturation in the presence of GSH-OEt increased the proportion of diploid blastocyst in aged cows. In contrast, GSH-OEt failed to enhance the oocyte maturation, fertilization, and embryo development in young cows. Taken together, the exogenous supplementation of GSH-OEt during IVM modulated the age-related oxidative damage of bovine oocytes and improved the developmental competence of oocytes in aged cows. Oocytes presented a distinct response to GSH-OEt treatment depending on the donor age. GSH-OEt supplementation during IVM could be of practical value through the efficiency improvement of chromosomally normal embryo production in aged cows.

A "Golden Age" for the discovery of new antileishmanial agents: Current status of leishmanicidal gold complexes and prospective targets beyond the trypanothione system.

Leishmaniasis is one of the most neglected diseases worldwide and is considered a serious public health issue. The current therapeutic options have several disadvantages that make the search for new therapeutics urgent. Gold compounds are emerging as promising candidates based on encouraging in vitro and limited in vivo results for several AuI and AuIII complexes. The antiparasitic mechanisms of these molecules remain only partially understood. However, a few studies have proposed the trypanothione redox system as a target, similar to the mammalian thioredoxin system, pointed out as the main target for several gold compounds with significant antitumor activity. In this review, we present the current status of the investigation and design of gold compounds directed at treating leishmaniasis. In addition, we explore potential targets in Leishmania parasites beyond the trypanothione system, taking into account previous studies and structure modulation performed for gold-based compounds.

Arabidopsis thaliana 3-mercaptopyruvate sulfurtransferases interact with and are protected by reducing systems.

The formation of a persulfide group (-SSH) on cysteine residues has gained attention as a reversible posttranslational modification contributing to protein regulation or protection. The widely distributed 3-mercaptopyruvate sulfurtransferases (MSTs) are implicated in the generation of persulfidated molecules and H2S biogenesis through transfer of a sulfane sulfur atom from a suitable donor to an acceptor. Arabidopsis has two MSTs, named STR1 and STR2, but they are poorly characterized. To learn more about these enzymes, we conducted a series of biochemical experiments including a variety of possible reducing systems. Our kinetic studies, which used a combination of sulfur donors and acceptors revealed that both MSTs use 3-mercaptopyruvate efficiently as a sulfur donor while thioredoxins, glutathione, and glutaredoxins all served as high-affinity sulfane sulfur acceptors. Using the redox-sensitive GFP (roGFP2) as a model acceptor protein, we showed that the persulfide-forming MSTs catalyze roGFP2 oxidation and more generally trans-persulfidation reactions. However, a preferential interaction with the thioredoxin system and glutathione was observed in case of competition between these sulfur acceptors. Moreover, we observed that MSTs are sensitive to overoxidation but are protected from an irreversible inactivation by their persulfide intermediate and subsequent reactivation by thioredoxins or glutathione. This work provides significant insights into Arabidopsis STR1 and STR2 catalytic properties and more specifically emphasizes the interaction with cellular reducing systems for the generation of H2S and glutathione persulfide and reactivation of an oxidatively modified form.

Bioactivation of trichloroethylene to three regioisomeric glutathione conjugates by liver fractions and recombinant human glutathione transferases: Species differences and implications for human risk assessment.

Enzymatic conjugation of glutathione (GSH) to trichloroethylene (TCE) followed by catabolism to the corresponding cysteine-conjugate, S-(dichlorovinyl)-L-cysteine (DCVC), and subsequent bioactivation by renal cysteine conjugate beta-lyases is considered to play an important role in the nephrotoxic effects observed in TCE-exposed rat and human. In this study, it is shown for the first time that three regioisomers of GSH-conjugates of TCE are formed by rat and human liver fractions, namely S-(1,2-trans-dichlorovinyl)-glutathione (1,2-trans-DCVG), S-(1,2-cis-dichlorovinyl)-glutathione (1,2-cis-DCVG) and S-(2,2-dichlorovinyl)-glutathione (2,2-DCVG). In incubations of TCE with rat liver fractions their amounts decreased in order of 1,2-cis-DCVG > 1,2-trans-DCVG > 2,2-DCVG. Human liver cytosol showed a more than 10-fold lower activity of GSH-conjugation, with amounts of regioisomers decreasing in order 2,2-DCVG > 1,2-trans-DCVG > 1,2-cis-DCVG. Incubations with recombinant human GSTs suggest that GSTA1-1 and GSTA2-2 play the most important role in human liver cytosol. GSTP1-1, which produces regioisomers in order 1,2-trans-DCVG > 2,2-cis-DCVG > 1,2-cis-DCVG, is likely to contribute to extrahepatic GSH-conjugation of TCE. Analysis of the products formed by a beta-lyase mimetic model showed that both 1,2-trans-DCVC and 1,2-cis-DCVC are converted to reactive products that form cross-links between the model nucleophile 4-(4-nitrobenzyl)-pyridine (NBP) and thiol-species. No NBP-alkylation was observed with 2,2-DCVC corresponding to its low cytotoxicity and mutagenicity. The lower activity of GSH-conjugation of TCE by human liver fractions, in combination with the lower fraction of potential nephrotoxic and mutagenic 1,2-DCVG-isomers, suggest that humans are at much lower risk for TCE-associated nephrotoxic effects than rats.

Glutathione conjugates of the mercapturic acid pathway and guanine adduct as biomarkers of exposure to CEES, a sulfur mustard analog.

Sulfur mustard (SM), a chemical warfare agent, is a strong alkylating compound that readily reacts with numerous biomolecules. The goal of the present work was to define and validate new biomarkers of exposure to SM that could be easily accessible in urine or plasma. Because investigations using SM are prohibited by the Organisation for the Prohibition of Chemical Weapons, we worked with 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of SM. We developed an ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) approach to the conjugate of CEES to glutathione and two of its metabolites: the cysteine and the N-acetylcysteine conjugates. The N7-guanine adduct of CEES (N7Gua-CEES) was also targeted. After synthesizing the specific biomarkers, a solid-phase extraction protocol and a UHPLC-MS/MS method with isotopic dilution were optimized. We were able to quantify N7Gua-CEES in the DNA of HaCaT keratinocytes and of explants of human skin exposed to CEES. N7Gua-CEES was also detected in the culture medium of these two models, together with the glutathione and the cysteine conjugates. In contrast, the N-acetylcysteine conjugate was not detected. The method was then applied to plasma from mice cutaneously exposed to CEES. All four markers could be detected. Our present results thus validate both the analytical technique and the biological relevance of new, easily quantifiable biomarkers of exposure to CEES. Because CEES behaves very similar to SM, the results are promising for application to this toxic of interest.