Modified immunoslotblot assay to detect hemi and sulfur mustard DNA adducts

Sulfur mustard (SM) is an old chemical warfare agent causing blisters (vesicant). Skin toxicity is thought to be partly caused by SM induced DNA damage. SM and the hemi mustard 2-chloroethyl ethyl sulfide (CEES) are bi- and monofunctional DNA alkylating agents, respectively. Both chemicals react especially with N₇ guanine. The most abundant adducts are 7-hydroxyethylthioethylguanine for SM (61%) and 7-ethyl thioethylguanine for CEES. Thus, DNA alkylation should serve as a biomarker of SM exposure. A specific monoclonal antibody (2F8) was previously developed to detect SM and CEES adducts at N₇ position by means of immunoslotblot (ISB) technique (van der Schans et al. (2004) [16]). Nitrogen mustards (HN-1, HN-2, HN-3) are alkylating agents with structural similarities, which can form DNA adducts with N₇ guanine. The aim of the presented work was to modify the van der Schans protocol for use in a field laboratory and to test the cross reactivity of the 2F8 antibody against nitrogen mustards. Briefly, human keratinocytes were exposed to SM and CEES (0–300 μM, 60 min) or HN-1, HN-2, HN-3 (120 min). After exposure, cells were scraped and DNA was isolated and normalized. 1 μg DNA was transferred to a nitrocellulose membrane using a slotblot technique. After incubation with 2F8 antibody, the DNA adducts were visualized with chromogen staining (3,3′-diaminobenzidine (DAB), SeramunGrün). Blots were photographed and signal intensity was quantified. In general, DAB was superior to SeramunGrün stain. A staining was seen from 30 nM to 300 μM of SM or CEES, respectively. However, statistically significant DNA adducts were detected after CEES and SM exposure above 30 μM which is below the vesicant threshold. No signal was observed after HN-1, HN-2, HN-3 exposure. The total hands-on time to complete the assay was about 36 h. Further studies are necessary to validate SM or CEES exposure in blister roofs of exposed patients.     

Fibrogenic cytokine levels in bronchoalveolar lavage aspirates 15 years after exposure to sulfur mustard

Over 100,000 Iranian war veterans suffer from chronic effects of mustard gas exposure. Sulfur mustard was used by Iraq during the Iraqi-imposed war on Iran (between 1980 and 1988). The major complaints of these patients are mild interstitial fibrosis and bronchiolitis. We aimed to determine the state of fibrosis progression and assessed transforming growth factor (TGF)-beta1 levels in pulmonary samples and in bronchoalveolar lavage (BAL) aspirates. A total of 126 war veterans confirmed for lung disease were assessed and compared with three control groups: 1) 64 veterans not exposed to chemical agents, 2) 12 idiopathic pulmonary fibrosis civilian patients, and 3) 33 normal persons. BAL was performed via a flexible fiber-optic bronchoscope and the standard manual method. Total protein was measured by Bradford assay, and samples were corrected with regard to coefficients. Samples were concentrated 15-fold by lyophilization and resolubilization. Samples were double-checked using an ELISA test kit. The Mann-Whitney test was used for the data analysis using commercial software. We detected that significant differences between TGF-beta1 levels between the case group and control group 1 (P = 0.001) and control group 3 (P = 0.003). No significant differences were found between the case group and control group 2 (P = 0.57). Inflammation and fibrotic processes in lung tissue of patients exposed to sulfur mustard may be progressive so IFN-gamma may be a useful drug to these patients' treatment.     

Mesenchymal stem cells are highly resistant to sulfur mustard

The effect of sulfur mustard (SM) to the direct injured tissues of the skin, eyes and airways is well investigated. Little is known about the effect of SM to mesenchymal stem cells (MSC). However, this is an interesting aspect. Comparing the clinical picture of SM it is known today that MSC play an important role e.g. in chronic impaired wound healing. Therefore we wanted to get an understanding about how SM affects MSC and if these findings might become useful to get a better understanding of the effect of sulfur mustard gas with respect to skin wounds.     

Silibinin as a potential therapeutic for sulfur mustard injuries

Sulfur mustard (SM) is a vesicating chemical warfare agent causing skin blistering, ulceration, impaired wound healing, prolonged hospitalization and permanent lesions. Silibinin, the lead compound from Silybum marianum, has also been discussed as a potential antidote to SM poisoning. However, its efficacy has been demonstrated only with regard to nitrogen mustards. Moreover, there are no data on the efficacy of the water-soluble prodrug silibinin-bis-succinat (silibinin-BS).     

Putative roles of inflammation in the dermatopathology of sulfur mustard

The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.     

Assessment of sulfur mustard interaction with basement membrane components

Bis-2-chloroethyl sulfide (sulfur mustard, HD) is a bifunctional alkylating agent which causes severe vesication characterized by slow wound healing. Our previous studies have shown that the vesicant HD disrupts the epidermal-dermal junction at the lamina lucida of the basement membrane. The purpose of this study was to examine whether HD directly modifies basement membrane components (BMCs), and to evaluate the effect of HD on the cell adhesive activity of BMCs. EHS laminin was incubated with [¹⁴C]HD, and extracted by gel filtration. Analysis of the [¹⁴C]HD-conjugated laminin fraction by a reduced sodium dodecyl sulfate-polyacrylaminde gel electrophoresis (SDS-PAGE) revealed the incorporation of radioactivity into both laminin subunits and a laminin trimer resistant to dissociation in reduced SDS-PAGE sample buffer, suggesting direct alkylation and cross-linking of EHS laminin by [¹⁴C]HD. Normal human foreskin epidermal keratinocytes were biosynthetically labeled with [³⁵S]cysteine.³⁵S-labeled laminin isoforms, Ae. B1e. B2e. laminin and K.B1e.B2e. laminin (using the nomenclature of Engel), fibronectin, and heparan sulfate proteoglycan were isolated by immunoprecipitation from the cell culture medium, treated with HD or ethanol as control, and then analyzed by SDS-PAGE. On reduced SDS gels, these three BMCs not treated with HD showed the typical profile of dissociated subunits. However, HD treatment caused the appearance of higher molecular weight bands indicative of cross-linking of subunits within these BMCs. The HD scavengers sodium thiosulfate and cysteine prevented the cross-linking of BMC subunits by HD. Finally, Tissue culture dishes coated with laminin or fibronectin were treated with HD or ethanol as a control, and human keratinocytes were plated on the BMC-coated surfaces. After 20 h of incubation, it was observed that cell adhesion was decreased significantly on the BMC-coated surfaces treated with HD. As expected, the preincubation of HD with cysteine diminished the HD inhibition of cell adhesion. Thus, HD alkylates adhesive macromolecules of the basement membrane zone and inhibits their cell adhesive activity. These findings support the hypothesis that the alkylation of basement membrane components by HD destabilizes the epidermal-dermal junction in the process of HD-induced vesication. The failure of the HD-alkylated BMCs to support the attachment of keratinocytes might also contribute to the slow reepithelialization of the wound site which is characteristic of HD-induced blistering.Abbreviations BMC basement membrane component - DEM Dulbecco's modified Eagle's medium - ECM extracellular matrix - EHS Englebreth-Holm-Swarm sarcoma - HD sulfur mustard - HSPG heparan sulfate proteoglycan - KGM keratinocyte growth medium - NHEK normal human keratinocytes - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Sulfur mustard induced nuclear translocation of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH)

Sulfur Mustard (SM) is a vesicant chemical warfare agent, which is acutely toxic to a variety of organ systems including skin, eyes, respiratory system and bone marrow. The underlying molecular pathomechanism was mainly attributed to the alkylating properties of SM. However, recent studies have revealed that cellular responses to SM exposure are of more complex nature and include increased protein expression and protein modifications that can be used as biomarkers. In order to confirm already known biomarkers, to detect potential new ones and to further elucidate the pathomechanism of SM, we conducted large-scale proteomic experiments based on a human keratinocyte cell line (HaCaT) exposed to SM. Surprisingly, our analysis identified glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as one of the up-regulated proteins after exposure of HaCaT cells to SM. In this paper we demonstrate the sulfur mustard induced nuclear translocation of GAPDH in HaCaT cells by 2D gel-electrophoresis (2D GE), immunocytochemistry (ICC), Western Blot (WB) and a combination thereof. 2D GE in combination with MALDI-TOF MS/MS analysis identified GAPDH as an up-regulated protein after SM exposure. Immunocytochemistry revealed a distinct nuclear translocation of GAPDH after exposure to 300 μM SM. This finding was confirmed by fractionated WB analysis. 2D GE and subsequent immunoblot staining of GAPDH demonstrated two different spot locations of GAPH (pI 7.0 and pI 8.5) that are related to cytosolic or nuclear GAPDH respectively. After exposure to 300 μM SM a significant increase of nuclear GAPDH at pI 8.5 occurred. Nuclear GAPDH has been associated with apoptosis, detection of structural DNA alterations, DNA repair and regulation of genomic integrity and telomere structure. The results of our study add new aspects to the pathophysiology of sulfur mustard toxicity, yet further studies will be necessary to reveal the specific function of nuclear GAPDH in the pathomechanism of sulfur mustard.     

The delayed effect of mustard gas on housekeeping gene expression in lung biopsy of chemical injuries

ObjectiveSulfur mustard (SM) was used as a chemical weapon in Iraq-Iran war. Exposed people have major complications in important organs such as pulmonary system. Some studies have shown that SM could affect the expression of endogenous genes and non-housekeeping genes, time dependently. To understand the accurate molecular mechanism of the delayed effect of SM, the identification of the gene expression pattern in these patients is essential. Hence, we have evaluated mRNA expression of four common housekeeping genes (ACTIN, PGK1, β2m, GAPDH) in SM-exposed and non-exposed (control) formalin-fixed, paraffin-embedded (FFPE) human lung tissues.MethodParaffin block of lung biopsy of SM-exposed people (11 cases) and people without exposure to SM as control group (9 cases) have been selected. The mRNA expression of four endogenous control genes has been evaluated by qRT-PCR. The stability value of each gene was calculated by different methods.ResultIt was found that ACTIN mRNA has the highest expression (30.26±2.87) and PGK1 has the lowest standard deviation (SD) (30.885±2.215) between pooled groups. The best correlation was between ACTIN and PGK1 expressions. The M value has shown that ACTIN and then PGK1 are the most stable housekeeping genes among. The results obtained from the GeNorm and NormFinder have indicated that the pair ACTIN- PGK1 is the most suitable choice for endogenous control genes.ConclusionACTIN and PGK1 genes are stable in studied lung tissues and are the better than two other housekeeping genes. In addition, mustard gas does not affect their expression in long term.     

L-Oxothiazolidine 4-carboxylate pretreatment of isolated human peripheral blood lymphocytes reduces sulfur mustard cytotoxicity

Sulfur mustard (HD, mustard gas) is a vesicant chemical warfare agent for which there is no specific medical countermeasure. A potential approach to combating the debilitating effects of this agent is the use of compounds that can react with this material before it interacts with critical macro-molecules. Glutathione (GSH), a tripeptide that exists in high concentrations in cells, reacts with HD and is involved in HD detoxification. Pretreatment of human peripheral blood lymphocytes (PBL) with 10 mmol/L L-oxothiazolidine-4-carboxylate (OTC), a "masked" cysteine precursor, increased intracellular glutathione levels 25-50% over control values. Pretreated PBL were harvested, washed, and exposed to 10, 50, or 100 µmol/L HD. Flow cytometry was used to measure cytotoxicity by propidium iodide uptake. Pretreatment of PBL with OTC led to small decreases in cytotoxicity after HD exposure. However, treatment of cells with OTC after HD exposure was not beneficial. Compounds that can modulate GSH levels within the cell may help to reduce the cytotoxicity of HD when used as a pretreatment.     

Biochemical manipulation of intracellular glutathione levels influences cytotoxicity to isolated human lymphocytes by sulfur mustard

Glutathione (GSH) is the major nonprotein thiol that can protect cells from damage due to electrophilic alkylating agents by forming conjugates with the agent. Sulfur mustard (HD) is an electrophilic alkylating agent that has potent mutagenic, carcinogenic, cytotoxic, and vesicant properties. Compounds that elevate or reduce intracellular levels of GSH may produce changes in cytotoxicity induced by sulfur mustard. Pretreatment of human peripheral blood lymphocytes (PBL) for 72 hr with 1 mM buthionine sulfoximine (BSO), which reduces intracellular GSH content to approximately 26% of control, appears to sensitize these in vitro cells to the cytotoxic effects of 10 M HD but not to higher HD concentrations. Pretreatment of PBL for 48 hr with 10 mM N-acetyl cysteine (NAC), which elevates intracellular glutathione levels to 122% of control, appears to partially protect these in vitro cells from the cytotoxic effects of 10 M HD but not to higher HD concentrations. Augmentation of intracellular levels of glutathione may provide partial protection against cytotoxicity of sulfur mustard.Abbreviations BSO L-buthionine (S,R)-sulfoximine - GSH glutathione - HD sulfur mustard - NAC N-acetyl-L-cysteine - PBL peripheral blood lymphocytes The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.     

Detoxification of sulfur mustard by enzyme-catalyzed oxidation using chloroperoxidase

One of the most interesting methods for the detoxification of sulfur mustard is enzyme-catalyzed oxidation. This study examined the oxidative destruction of a sulfur mustard by the enzyme chloroperoxidase (EC 1.11.1.10). Chloroperoxidase (CPO) belongs to a group of enzymes that catalyze the oxidation of various organic compounds by peroxide in the presence of a halide ion. The enzymatic oxidation reaction is affected by several factors: pH, presence or absence of chloride ion, temperature, the concentrations of hydrogen peroxide and enzyme and aqueous solubility of the substrate. The optimum reaction conditions were determined by analyzing the effects of all factors, and the following conditions were selected: solvent, Britton–Robinson buffer (pH = 3) with tert-butanol (70:30 v/v); CPO concentration, 16 U/mL; hydrogen peroxide concentration, 40 mmol/L; sodium chloride concentration, 20 mmol/L. Under these reaction conditions, the rate constant for the reaction is 0.006 s⁻¹. The Michaelis constant, a measure of the affinity of an enzyme for a particular substrate, is 1.87 × 10⁻³ M for this system. The Michaelis constant for enzymes with a high affinity for their substrate is in the range of 10⁻⁵ to 10⁻⁴ M, so this value indicates that CPO does not have a very high affinity for sulfur mustard.     

NMR spectroscopy-based metabolomic study of serum in sulfur mustard exposed patients with lung disease

Sulfur mustard (SM) is a vesication chemical warfare agent for which there is currently no antidote. Despite years of research, there is no common consensus about the pathophysiological basis of chronic pulmonary disease caused by this chemical warfare agent. In this study, we combined chemometric techniques with nuclear magnetic resonance (NMR) spectroscopy to explore the metabolic profile of sera from SM-exposed patients. A total of 29 serum samples obtained from 17 SM-injured patients, and 12 healthy controls were analyzed by Random Forest. Increased concentrations of seven amino acids, glycerol, dimethylamine, ketone bodies, lactate, acetate, citrulline and creatine together with the decreased very low-density lipoproteins (VLDL) levels were observed in patients compared with control subjects. Our study reveals the metabolic profile of sera from SM-injured patients and indicates that NMR-based methods can distinguish these patients from healthy controls.     

Effect of sulfur exposure on protease activity in human peripheral blood lymphocytes

Sulfur mustard is a waemical warfare blistering agent for which neither the mechanism of action nor an antidote is known. Papirmeister et al. (1985) have postulated a biochemical hypothesis for mustard-induced cutaneous injury involving a sequelae of DNA alkylation, metabolic disruption and activation of protease. Human peripheral blood lymphocytes in cell cultures were employed as an in vitro model for alkylating agent toxicity. A chromogenic peptide substrate assay was used for detection of protease in lymphocytes treated with sulfur mustard or chloroethyl ethyl sulfide. Exposure of human peripheral blood lymphocytes from normal donors to these alkylating agents resulted in an increase in cell associated protease activity. This increase in protease activity may contribute to the pathology or act as an indicator to predict methods of therapeutic intervention for sulfur mustard toxicity.Abbreviations PBL peripheral blood lymphocytes - CEES chloroethyl ethyl sulfide - DFP diisopropyl fluoro-phosphate - pNA p-nitroaniline - CPSPA Chromogenic Peptide Substrate Protease Assay The opinions or assertions herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.     

The relationship between ACE inhibitory activity and degradations of sulfur containing materials in Dolsan leaf mustard juice

This study was carried out to investigate the relationship ACE inhibitory activity and degradations of sulfur containing materials in Dolsan leaf mustard juice (DLMJ). The changes of sulfur containing materials which were treated with autolysis, myrosinase, ascorbate and papain were studied, as well as the changes of ACE inhibitory activity in DLMJ. At 37°C, sulfur containing materials by autolysis decreased most rapidly from 0.43% to 0.13% in the second day. Conversely, ACE inhibitory activity increased most from 66% to 87%, in the second day at 37°C. As myrosinase concentrations increased more, sulfur containing materials in DLMJ decreased more. The ACE inhibitory activities at 0, 0.5, 1, 2, and 4 Units of myrosinase for 240 min later were 70, 74, 75, 82, and 85%, respectively. At 1 mM ascorbate, concentrations of sulfur containing materials in DLMJ decreased more significantly on the second day than on the other days. At 1 mM ascorbate for 6 days, ACE inhibitory activity reached a maximum of about 92%. And, an increase of papain concentration was noted in accordance with a decreased sulfur containing materials. The maximum rate of ACE inhibitory activity at control, 3, 6, and 12 Units of papains treatments was shown as 70, 70, 75, and 78% at 60 min, respectively. These results suggested that the degradation of sulfur containing materials led to the increase of ACE inhibitory activity. Consequently, it was suggested that ACE inhibiting was significantly related to the degradatives of sulfur containing materials.     

Unconventional cytokine profiles and development of T cell memory in long-term survivors after cancer vaccination

Cancer vaccine trials frequently report on immunological responses, without any clinical benefit. This paradox may reflect the challenge of discriminating between effective and pointless immune responses and sparse knowledge on their long-term development. Here, we have analyzed T cell responses in long-term survivors after peptide vaccination. There were three main study aims: (1) to characterize the immune response in patients with a possible clinical benefit. (2) To analyze the long-term development of responses and effects of booster vaccination. (3) To investigate whether the Th1/Th2-delineation applies to cancer vaccine responses. T cell clones were generated from all nine patients studied. We find that surviving patients harbor durable tumor-specific responses against vaccine antigens from telomerase, RAS or TGFβ receptor II. Analyses of consecutive samples suggest that booster vaccination is required to induce robust T cell memory. The responses exhibit several features of possible clinical advantage, including combined T-helper and cytotoxic functionality, recognition of naturally processed antigens and diverse HLA-restriction and fine-specificity. CD4⁻CD8⁻ T cell clones display unconventional cytotoxicity and specifically kill tumor cells expressing mutated TGFβ receptor II. Cytokine profiling on the long-term survivors demonstrates high IFNγ/IL10-ratios, favoring immunity over tolerance, and secretion of multiple chemokines likely to mobilize the innate and adaptive immune system. Interestingly, these pro-inflammatory cytokine profiles do not follow a Th1/Th2-delineation. Most IFNγ^high/IL4^low/IL10^low cultures include high concentrations of hallmark Th2-cytokines IL-5 and IL-13. This does not reflect a mixture of Th1- and Th2-clones, but applies to 19/20 T cell clones confirmed to be monoclonal through TCR clonotype mapping. The present study identifies several factors that may promote clinical efficacy and suggests that cytokine profiling should not rely on the Th1/Th2-paradigm, but assess the overall inflammatory milieu and the balance between key cytokines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

NMR- and GC/MS-based metabolomics of sulfur mustard exposed individuals: a pilot study

Sulfur mustard (SM) is a potent alkylating agent and its effects on cells and tissues are varied and complex. Due to limitations in the diagnostics of sulfur mustard exposed individuals (SMEIs) by noninvasive approaches, there is a great necessity to develop novel techniques and biomarkers for this condition. We present here the first nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC/MS) metabolic profiling of serum from and healthy controls to identify novel biomarkers in blood serum for better diagnostics. Of note, SMEIs were exposed to SM 30 years ago and that differences between two groups could still be found. Pathways in which differences between SMEIs and healthy controls are observed are related to lipid metabolism, ketogenesis, tricarboxylic acid (TCA) cycle and amino acid metabolism.