Mustard Analog Research Articles

Mesenchymal Stem Cell Therapy Mitigates Acute and Chronic Lung Damages of Sulfur Mustard Analog Exposure.

Sulfur mustard (SM) is an established chemical weapon that can result in severe damage to parts of the body. Currently, there are no effective treatments available for SM-caused damage. We aimed to investigate the therapeutic potential of adipose-derived mesenchymal stromal cells (AD-MSCs) and conditioned medium (CM-MSCs) in acute and chronic pulmonary mouse models caused by 2-chloroethyl ethyl sulfide (CEES), an SM analog. The mice were divided into 4 experimental groups:(1) CEES+AD-MSCs, (2) CEES+CM-MSCs, (3) CEES, and (4) control. The model observation time was divided into 7 days for the short and 6 months for the long term. AD-MSCs were injected into mice via intraperitoneal injection 24 hours after CEES exposure. The therapeutic effects of AD-MSCs on pulmonary tissue damage were assessed using histopathologic assay, measuring the neutrophil count, and bronchial alveolar lavage fluid (BALF) protein level. The levels of inflammatory and anti-inflammatory cytokines were evaluated using the enzyme-linked immunosorbent assay as the outcomes of interest. Lung damage progression was reduced by AD-MSC treatment in mice after CEES injection into the peritoneum. The proportion of CD11b+F4/80+ macrophages in peritoneum was significantly lowered by AD-MSC treatment following CEES exposure. AD-MSC administration also reduced the level of pro-inflammatory cytokines, BALF protein, and nitric oxide levels in the peritoneal cavity. By reducing inflammation and enhancing tissue healing, AD-MSCs and CM-MSC help prevent acute lung damage caused by CEES. The current study supports the use of a mouse model as a solid experimental foundation and indicates potential use for future cell treatment.

The blistering warfare agent O-mustard (agent T) generates protein-adducts with human serum albumin useful for biomedical verification of exposure and forms intramolecular cross-links.

The highly blistering sulfur mustard analogue agent T (bis(2-chloroethylthioethyl) ether), also known as O-mustard or oxy-mustard, is a common impurity in military grade sulfur mustard (SM) and a component of mixtures such as "HT" that are still found in old munitions. Together with sesquimustard (Q), it is the most important SM analogue and tightly regulated as a Schedule 1 chemical under the Chemical Weapons Convention. We report the adducts of T with nucleophilic Cys34 and other residues in human serum albumin (HSA) formed in vitro. A micro liquid chromatography electrospray ionization high-resolution tandem-mass spectrometry method (µLC-ESI MS/HRMS) was developed for the detection and identification of biomarker peptides alkylated by a T-derived hydroxyethylthioethyloxyethylthioethyl (HETEOETE)-moiety (as indicated by an asterisk below). Following proteolysis of T-exposed human plasma with pronase, the dipeptide Cys34*Pro and the single amino acid residue His* were produced. The use of proteinaseK yielded Cys34*ProPhe and theuse of pepsin generated ValThrGlu48*Phe, AlaGlu230*ValSerLysLeu, and LeuGlyMet329*Phe. Corresponding peptide-adducts of SM and Q were detected in a common workflow that in principle allowed the estimation of the mustard or mustard composition encountered during exposure. Novel adducts of Q at the Glu230 and Met239 residues were detected and are reported accordingly. Based on molecular dynamics simulations, we identified regular interactions of the Cys34(-HETEOETE)-moiety with several glutamic acid residues in HSA including Glu86, which is not an obvious interaction partner by visual inspection of the HSA crystal structure. The existence of this and other intramolecular cross-links was experimentally proven for the first time.

Assessing the risk of tumor lysis syndrome associated with the use of antineoplastic agents: a real-world pharmacovigilance study based on the FDA Adverse Event Reporting System database.

The use of antineoplastic agents is one of the important triggers of tumor lysis syndrome (TLS), but there is still a lack of comprehensive understanding of antineoplastic agents that may trigger TLS and the TLS risk differences between different antineoplastic agents. This study aims to investigate the TLS risk of different antineoplastic agents and provide reference information for clinical practice. Real-world adverse events data in the FDA Adverse Event Reporting System (FAERS) database were used as the basis for the disproportionality analysis. We reviewed the TLS reports in the FAERS database from 2004 to 2022 to summarize an antineoplastic agent list that was reported to trigger TLS, based on which we conducted disproportionality analysis to assess the TLS risk of each antineoplastic agent. In all, 164 antineoplastic agents were reported to trigger TLS. On the whole, rituximab was the most reported antineoplastic agent in TLS reports, followed by cyclophosphamide, venetoclax, doxorubicin, and etoposide, while tagraxofusp was the antineoplastic agent with the highest adverse drug reaction (ADR) signal strength in signal detection, followed by floxuridine, pentostatin, tebentafusp, and venetoclax. Integrating ADR signal detection results, 129 of 164 antineoplastic agents showed at least one positive ADR signal, and six antineoplastic agents (bevacizumab, carboplatin, cisplatin, fluorouracil, lenvatinib, and paclitaxel) have the highest total number of positive signals. Further classifying the 164 antineoplastic agents into 46 chemical subgroups to conduct ADR signal detection, nitrogen mustard analogs were the most reported antineoplastic agent subclasses, followed by clusters of differentiation 20 inhibitors, and pyrimidine analogs, while clusters of differentiation 22 inhibitors were the antineoplastic agent subclass with the highest ADR signal strength, followed by podophyllotoxin derivatives and actinomycines. Our study showed the TLS risk characteristics of 164 antineoplastic agents by detecting and integrating ADR signals, which may help to optimize clinical practice.

Life-Cycle-Dependent Toxicities of Mono- and Bifunctional Alkylating Agents in the 3R-Compliant Model Organism C. elegans.

Caenorhabditis elegans (C. elegans) is gaining recognition and importance as an organismic model for toxicity testing in line with the 3Rs principle (replace, reduce, refine). In this study, we explored the use of C. elegans to examine the toxicities of alkylating sulphur mustard analogues, specifically the monofunctional agent 2-chloroethyl-ethyl sulphide (CEES) and the bifunctional, crosslinking agent mechlorethamine (HN2). We exposed wild-type worms at different life cycle stages (from larvae L1 to adulthood day 10) to CEES or HN2 and scored their viability 24 h later. The susceptibility of C. elegans to CEES and HN2 paralleled that of human cells, with HN2 exhibiting higher toxicity than CEES, reflected in LC50 values in the high µM to low mM range. Importantly, the effects were dependent on the worms' developmental stage as well as organismic age: the highest susceptibility was observed in L1, whereas the lowest was observed in L4 worms. In adult worms, susceptibility to alkylating agents increased with advanced age, especially to HN2. To examine reproductive effects, L4 worms were exposed to CEES and HN2, and both the offspring and the percentage of unhatched eggs were assessed. Moreover, germline apoptosis was assessed by using ced-1p::GFP (MD701) worms. In contrast to concentrations that elicited low toxicities to L4 worms, CEES and HN2 were highly toxic to germline cells, manifesting as increased germline apoptosis as well as reduced offspring number and percentage of eggs hatched. Again, HN2 exhibited stronger effects than CEES. Compound specificity was also evident in toxicities to dopaminergic neurons-HN2 exposure affected expression of dopamine transporter DAT-1 (strain BY200) at lower concentrations than CEES, suggesting a higher neurotoxic effect. Mechanistically, nicotinamide adenine dinucleotide (NAD+) has been linked to mustard agent toxicities. Therefore, the NAD+-dependent system was investigated in the response to CEES and HN2 treatment. Overall NAD+ levels in worm extracts were revealed to be largely resistant to mustard exposure except for high concentrations, which lowered the NAD+ levels in L4 worms 24 h post-treatment. Interestingly, however, mutant worms lacking components of NAD+-dependent pathways involved in genome maintenance, namely pme-2, parg-2, and sirt-2.1 showed a higher and compound-specific susceptibility, indicating an active role of NAD+ in genotoxic stress response. In conclusion, the present results demonstrate that C. elegans represents an attractive model to study the toxicology of alkylating agents, which supports its use in mechanistic as well as intervention studies with major strength in the possibility to analyze toxicities at different life cycle stages.

HSP90/CDC37 inactivation promotes degradation of LKB1 protein to suppress AMPK signaling in bronchial epithelial cells exposed to sulfur mustard analog, 2-chloroethyl ethyl sulfide

To investigate the role of the liver kinase (LK) B1 protein, an activator of AMP-activated protein kinase (AMPK), in AMPK signaling suppression when exposed to vesicant, a kind of chemical warfare agent. Cultured human bronchial epithelial cells were inflicted with sulfur mustard (SM) analog, 2-chloroethyl ethyl sulfide (CEES) of 0.2–1.0 mM concentration, and cell proliferation, apoptosis, autophagy, and cellular ATP level were analyzed up to 24 h after the exposure. Focusing on LKB1, heat shock protein (HSP) 90, and cell division cycle (CDC) 37 proteins, the protein expression, phosphorylation, and interaction were examined with western blot, immunofluorescence staining, and/or immunoprecipitation. AMPK signaling was found to be inhibited 24 h after being exposed to either sub-cytotoxic (0.5 mM) or cytotoxic (1.0 mM) concentration of CEES based on MTS assay. Consistently, the degradation of the LKB1 protein and its less interaction with the HSP90/CDC37 complex was confirmed. It was found that 1.0, not 0.5 mM CEES also decreased the CDC37 protein, proteasome activity, and cellular ATP content that modulates HSP90 protein conformation. Inhibiting proteasome activity could alternatively activate autophagy. Finally, either 0.5 or 1.0 mM CEES activated HSP70 and autophagy, and the application of an HSP70 inhibitor blocked autophagy and autophagic degradation of the LKB1 protein. In conclusion, we reported here that AMPK signaling inactivation by CEES was a result of LKB1 protein loss via less protein complex formation and enhanced degradation.

Evaluation of the effects of dexamethasone in modulating breathing pattern decomplexification in rats with 2-chloroethyl ethyl sulfide-induced lung injury

"Background and Objectives: Sulfur mustard is a functional alkylating chemical warfare agent that gives rise to appalling lung injury. In people with pulmonary diseases, including asthma, the complication of respirational dynamics is reduced. However, the complexity of breathing patterns in lung injury caused by chemical agents is not clear. In the current study, the outcome of 2-chloroethyl ethyl sulfide (CEES), and mustard analogue, upon breathing pattern of rats without or with treatment were reviewed. Methods: The interbeat interval (IBI) and respiratory volume (RV) data have been acquired from spontaneous respiration rats with lung injury by CEES using a whole-body plethysmograph. We calculated mean and coefficient of variation, alpha exponent derived from detrended fluctuation analysis (DFA), and sample entropy of IBI and RV. Finding: Entropy examination of respiratory variation displayed reduced inconsistency (less complication) in the breathing pattern of this rat model of lung injury. The mustard analogue also led to increased lung inflammation in damaged rats. However, treatment by NAC and dexamethasone had a compelling impact on the complication of the breathing rhythm and lung inflammation of rats with lung injury. Conclusion: Our findings show that inflammation could be the possible origin of respiratory dynamics shifting apart from the normal variation in CEES-induced lung injury"

Sulfur mustard analog 2-chloroethyl ethyl sulfide increases triglycerides by activating DGAT1-dependent biogenesis and inhibiting PGC1ɑ-dependent fat catabolism in immortalized human bronchial epithelial cells

Using sulfur mustard analog 2-chloroethyl ethyl sulfide (CEES), we established an in vitro model by poisoning cultured immortalized human bronchial epithelial cells. Nile Red staining revealed lipids accumulated 24 h after a toxic dose of CEES (0.9 mM). Lipidomics analysis showed most of the increased lipids were triglycerides (TGs), and the increase in TGs was further confirmed using a Triglyceride-Glo™ Assay kit. Protein and mRNA levels of DGAT1, an important TG biogenesis enzyme, were increased following 0.4 mM CEES exposure. Under higher dose CEES (0.9 mM) exposure, protein and mRNA levels of PPARγ coactivator-1ɑ (PGC-1ɑ), a well-known transcription factor that regulates fatty acid oxidation, were decreased. Finally, application with DGAT1 inhibitor A 922500 or PGC1ɑ agonist ZLN005 was able to block the CEES-induced TGs increase. Overall, our dissection of CEES-induced TGs accumulation provides new insight into energy metabolism dysfunction upon vesicant exposure. HIGHLIGHTS In CEES (0.9 mM)-injured cells: Triglycerides (TGs) were abundant in the accumulated lipids. Expression of DGAT1, not DGAT2, was increased. Expression of PGC1ɑ, not PGC1β, was reduced. DGAT1 inhibitor or PGC1ɑ agonist blocked the CEES-mediated increase in TGs.

Bendamustine: A review of pharmacology, clinical use and immunological effects (Review).

Bendamustine is an alkylating agent classified into the group of nitrogen mustard analogues, synthesized almost sixty years ago. It was registered in former East Germany in 1971 and approved by the US Food and Drug Administration in 2008 for treatment of chronic lymphocytic leukemia and indolent B-cell non-Hodgkin lymphoma. Considering its beneficial properties in the therapy of relapsed or refractory hematological malignancies, synergistic effects with other antineoplastic agents and increasing recent reports on its immunomodulatory effects, bendamustine has once again gained its justified attention. The uniqueness of bendamustine-mediated effects should be observed keeping in mind its distinctive structure with structural similarities to both alkylating agents and purine analogs. In the present review, the current knowledge on the use of bendamustine in oncology, its pharmacokinetics, mechanism of action and toxicity was summarized. In addition, its immune-modulating effects that have not been fully elucidated so far are emphasized, hoping to encourage further investigations of this unique drug.

MicroPET imaging of bacterial infection with nitroreductase-specific responsive 18F-labelled nitrogen mustard analogues.

Bacterial infection and antibiotic resistance are serious threats to human health. This study aimed to develop two novel radiotracers, 18F-NTRP and 18F-NCRP, that possess a specific nitroreductase (NTR) response to image deep-seated bacterial infections using positron emission tomography (PET). This method can distinguish infection from sterile inflammation. 18F-NTRP and 18F-NCRP were synthesized via a one-step method; all the steps usually involved in tracer radiosynthesis were successfully adapted in the All-In-One automated module. After the physiochemical properties of 18F-NTRP and 18F-NCRP were characterized, their specificity and selectivity for NTR were verified in E. coli and S. aureus. The ex vivo biodistribution of the tracers was evaluated in normal mice. MicroPET-CT imaging was performed in mouse models of bacterial infection and inflammation after the administration of 18F-NTRP or 18F-NCRP. Fully automated radiosynthesis of 18F-NTRP and 18F-NCRP was achieved within 90-110min with overall decay-uncorrected, isolated radiochemical yields of 21.24 ± 4.25% and 11.3 ± 3.78%, respectively. The molar activities of 18F-NTRP and 18F-NCRP were 320 ± 40GBq/μmol and 275 ± 33GBq/µmol, respectively. In addition, 18F-NTRP and 18F-NCRP exhibited high selectivity and specificity for NTR response. PET-CT imaging in bacteria-infected mouse models with 18F-NTRP or 18F-NCRP showed significant radioactivity uptake in either E. coli- or S. aureus-infected muscles. The uptake for E. coli-infected muscles, 2.4 ± 0.2%ID/g with 18F-NTRP and 4.05 ± 0.49%ID/g with 18F-NCRP, was up to three times greater than that for uninfected control muscles. Furthermore, for both 18F-NTRP and 18F-NCRP, the uptake in bacterial infection was 2.6 times higher than that in sterile inflammation, allowing an effective distinction of infection from inflammation. 18F-NTRP and 18F-NCRP are worth further investigation to verify their potential clinical application for distinguishing bacterial infection from sterile inflammation via their specific NTR responsiveness.

Evidence for the systemic diffusion of (2-chloroethyl)-ethyl-sulfide, a sulfur mustard analog, and its deleterious effects in brain

Sulfur mustard, a chemical warfare agent known to be a vesicant of skin, readily diffuses in the blood stream and reaches internal organs. In the present study, we used the analog (2-chloroethyl)-ethyl-sulfide (CEES) to provide novel data on the systemic diffusion of vesicants and on their ability to induce brain damage, which result in neurological disorders. SKH-1 hairless mice were topically exposed to CEES and sacrificed at different time until 14 days after exposure. A plasma metabolomics study showed a strong systemic impact following a self-protection mechanism to alleviate the injury of CEES exposure. This result was confirmed by the quantification of specific biomarkers in plasma. Those were the conjugates of CEES with glutathione (GSH-CEES), cysteine (Cys-CEES) and N-acetyl-cysteine (NAC-CEES), as well as the guanine adduct (N7Gua-CEES). In brain, N7Gua-CEES could be detected both in DNA and in organ extracts. Similarly, GSH-CEES, Cys-CEES and NAC-CEES were present in the extracts until day14. Altogether, these results, based on novel exposure markers, confirm the ability of vesicants to induce internal damage following dermal exposure. The observation of alkylation damage to glutathione and DNA in brain provides an additional mechanism to the neurological insult of SM.

Small-interfering RNA for c-Jun attenuates cell death by preventing JNK-dependent PARP1 cleavage and DNA fragmentation in nitrogen mustard-injured immortalized human bronchial epithelial cells.

Sulfur mustard (a type of vesicant) can directly damage lung bronchial epithelium via aerosol inhalation, and prevalent cell death is an early event that obstructs the respiratory tract. JNK/c-Jun is a stress response pathway, but its role in cell death of the injured cells is not clear. Here, we report that JNK/c-Jun was activated in immortalized human bronchial epithelial (HBE) cells exposed to a lethal dose (20μM) of nitrogen mustard (NM, a sulfur mustard analog). c-Jun silencing using small-interfering RNA (siRNA) rendered the cells resistant to NM-mediated cell death by blocking poly(ADP-ribose) polymerase 1 (PARP1) cleavage and DNA fragmentation. In addition, the transduction of upstream extrinsic (Fasl-Fas-caspase-8) and intrinsic (loss of Bcl-2 and mitochondrial membrane potential, ΔΨm) apoptosis pathways, as well as phosphorylated (p)-H2AX (Ser139), an epigenetic marker contributing to DNA fragmentation and PARP1 activity, was partially suppressed. To mimic the detachment of cells by NM, HBE cells were trypsinized and seeded on culture plates that were pre-coated with poly-HEMA to prevent cell adhesion. The JNK/c-Jun pathway was found to be activated in the detached cells. In conclusion, our results indicate that JNK/c-Jun pathway activation is necessary for NM-caused HBE cell death and further suggest that c-Jun silencing may be a potential approach to protect HBE cells from vesicant damage.

Mass spectrometric analysis of adducts of sulfur mustard analogues to human plasma proteins: approach towards chemical provenancing in biomedical samples.

The primary aim of this study was to identify biomarkers of exposure to some so-called Schedule 1 sulfur mustard (HD) analogues, in order to facilitate and expedite their retrospective analysis in case of alleged use of such compounds. Since these HD analogues can be regarded as model compounds for possible impurities of HD formed during synthesis processes, the secondary aim was to explore to which extent these biomarkers can be used for chemical provenancing of HD in case biomedical samples are available. While the use of chemical attribution signatures (CAS) for neat chemicals or for environmental samples has been addressed quite frequently, the use of CAS for investigating impurities in biomedical samples has been addressed only scarcely. Human plasma was exposed to each of the five HD analogues. After pronase or proteinase K digestion of precipitated protein and sample work-up, the histidine (His) and tripeptide (CPF) adducts to proteins were analyzed, respectively. Adducts of the analogues could still be unambiguously identified next to the main HD adducts in processed plasma samples after exposure to HD mixed with each of the analogues, at a 1% level relative to HD. In conclusion, we have identified plasma protein adducts of a number of HD analogues, which can be used as biomarkers to assess an exposure to these Schedule 1 chemicals. We have shown that adducts of these analogues can still be analyzed after work-up of plasma samples which had been exposed to these analogues in a mixture with HD, supporting the hypothesis that biomedical sample analysis might be useful for chemical provenancing.

Immunomodulatory Effect of Curcumin in the Upregulation of Inflammasome Pathway Genes Induced by Sulfur Mustard Analog: An In-vitro Study

Sulfur Mustard (SM) induces cell injury via exerting oxidative stress, protease-anti protease imbalance, and inflammation. Inflammasome as one part of innate immunity has a critical role in the recognition of cell injuries and the initiation of the inflammation process by releasing IL-1β. Hence, the present study investigated the effects of the sub-lethal doses of 2-chloroethyl ethyl sulfide (CEES) as SM analog on the gene expression level of inflammasome-related genes as well as the potential protective effects of curcumin on this process. The effects of sub-lethal doses (500, 1000, and 2500 mM) of CEES on pulmonary epithelial cell line (A549) were determined at various time points (12, 24, and 48 h). Following the treatment of cells with CEES, the kinetic alterations of the expression levels of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB1), NLR family pyrin domain containing 1 (NLRP1), Caspase-1 (Casp1), and Interleukin-1β (IL-1β) genes were analyzed; using real-time PCR. In addition, the concurrent protective effects of different doses of curcumin (20, 40, 80, and 160 mM) on modulating the effects of CEES were studied. Although it was found that the lowest sub-lethal dose of CEES (500 mM) was able to up-regulate the inflammasome-related genes, the maximum alterations occurred 48 h after the treatment with the higher sub-lethal dose (2500 mM) of CEES. The maximum alteration occurred in Casp1 (38 fold), IL-1β (19 fold), and NLRP1 (~4 fold) genes (p<0.0001). However, the NF-ĸB gene expression level did not alter following CEES exposure. Even though low doses of curcumin (20, 40, and 80 mM) were able to down-regulate the studied genes, it was found that the treatment of cells with 160 mM of curcumin for 48 h was able to normalize the expression level of all genes. The present study concludes that curcumin as an anti-inflammatory agent may have beneficial immunomodulatory effects following CEES exposure.

Temperature‐responsive voltammetric sensor based on stimuli-sensitive semi-interpenetrating polymer network conductive microgels for reversible switch detection of nitrogen mustard analog chlorambucil (Leukeran™)

In this study, we report the synthesis of thermosensitive poly(N-vinylcaprolactam) semi-interpenetrated conductive polypyrrole microgels (PVCL/PPY semi-IPN MGs) for “on-off” switchable electrochemical sensing of carcinogenic nitrogen mustard drug chlorambucil (Leukeran™). The synthesized PVCL/PPY semi-IPN MGs modified electrode acts as a thermo-reversible switch-like sensor for the electrochemical oxidation of chlorambucil due to the temperature stimulus-response of PVCL. Cyclic voltammetry (CV) of chlorambucil at the PVCL/PPY semi-IPN MGs modified electrode manifest well-defined higher oxidation peak current when the electrolyte temperature is above the TLCST of PVCL, signifying the “on” state. This chlorambucil electro-oxidation peak current reduced below the TLCST of PVCL, signifying the “off” state. Repeatable “on-off” switching of the CV responses of chlorambucil at the PVCL/PPY semi-IPN MGs modified electrode was attained by controlling the electrolyte temperature. The proposed temperature-responsive switched sensor had a wide dynamic range (0.02 to 420 µM), excellent sensitivity, and a low detection limit of 1.98 nM for chlorambucil at 40 °C compared to 25 °C. Interestingly, the proposed sensor proves excellent selectivity for detecting chlorambucil in potential interference compounds. Moreover, the proposed method was successfully used to detect chlorambucil in human blood serum and urine samples.

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.

The immunomodulatory effects of mesenchymal stem cells on long term pulmonary complications in an animal model exposed to a sulfur mustard analog

IntroductionSulfur Mustard (SM) is one of the most lethal chemicals with major complications manifested in the lungs. Although the pathogenesis behind SM-induced lung injury still remains poorly understood, prolonged activation and the imbalance of two major macrophage populations (M1 and M2) have been suggested to be involved. Here, we tried to investigate the effectiveness of adipose-derived mesenchymal stem cells (AD-MSC) on long-term lesions induced by CEES, an SM analog. The modulation of pulmonary immune cells and alveolar macrophage phenotype alteration was studied in the animal model used. MethodsHistopathological changes were investigated in the lungs and analysis of surface markers of alveolar macrophages as well as their cytokine expression in the BAL fluid was carried out by flow cytometry and ELISA, respectively. ResultsTreatment of mice with AD-MSC after intraperitoneal administration of CEES (10 mg/kg) reduces progressive histopathologic changes in the lung. Flow cytometric analysis of isolated alveolar macrophages in the bronchoalveolar lavage showed that the accumulation of both M1 and M2 macrophages in response to CEES was reduced by MSC administration. AD-MSCs caused a marked reduction in the CD86- and CD206-expressing macrophages compared to the untreated groups. The modulating effect of AD-MSCs in the M1-subset was much more significant compared to M2.These findings suggest that AD-MSCs understand their environment and restore the balance in disorders associated with Th1 or Th2 imbalance. Our results indicate that MSCs may represent an effective approach to repair lung injury induced by mustards.

Time course study of oxidative stress in sulfur mustard analog 2‑chloroethyl ethyl sulfide-induced toxicity

Oxidative stress is the major mechanism impairing cell homeostasis, inducing cell death and tissue damage in sulfur mustard (SM)-exposed individuals. The aim of the present study was to evaluate time course changes of oxidative stress in the mice exposed with 2‑chloroethyl ethyl sulfide (CEES) as SM analog. For this purpose, male BALB/c mice were divided into control groups and experimental groups that received CEES (10 mg/kg) through intraperitoneal injection. In both groups, animals were euthanized at three periods: short (12, 24 h and 1 week), medium (1, 2 and 3 months) and long-term (5 and 6 months) after CEES exposure. Oxidative stress indices and the antioxidant defense systems were evaluated in lung and liver tissues. The time course findings in both tissues showed a significant increase in oxidative damage markers such as malondialdehyde (lung P < 0.001, liver P < 0.001), protein carbonyl (lung P < 0.0001), and 8-hydroxy-deoxyguanosine (lung P < 0.0001, Liver P < 0.0001) and also a significant reduction in the antioxidant defense system including reduced glutathione level (lung P < 0.001, Liver P < 0.001,), activities of catalase (lung P < 0.01 and liver P < 0.05), superoxide dismutase (lung P < 0.05), glutathione S‑transferase (lung P < 0.05, liver P < 0.01), glutathione peroxidase (lung, P < 0.05, Liver P < 0.05) and glutathione reductase (lung P < 0.001, liver P < 0.01) in the long-term. However, these changes occur with less intensity in the short-term and return to the normal status in the medium-term. Moreover, there was a positive time course correlation between oxidative damage indices and the percent of histopathological damage in both tissues (P < 0.05). This correlation finding confirms and supports the fact that time course oxidative-antioxidant imbalance plays an important role in the development of SM-induced acute and delayed injuries.

Phospholipid and Triglyceride keep vortex-dispersed sulfur mustard analog as undissolved droplets which induce necrosis from nearby cells via declining mitochondrial membrane potential

SM (Sulfur mustard) is an oily, hydrophobic, and lipophilic chemical agent that damages cells with intricate patterns. CEES (2-chloroethyl ethyl sulfide soluble) is a standard SM analog commonly employed in the toxicity mechanism study. To obtain ideal results in vitro, researchers should disperse CEES well in the medium, avoiding the presence of undissolved droplets. However, such a purpose is not easy to reach under the conventional solution preparation, and the information about droplet formation and function is little available. Here, we showed that phospholipid and triglyceride, two essential components of serum lipids, could prevent CEES from dissolving in water after vortex, which kept partial CEES as small droplets. By detecting CEES level, we proved that residual droplets slowed CEES hydrolysis and conversion. Under the microscope, CEES droplets were observed to degrade and diffuse with time to induce the necrosis and mitochondrial membrane potential decline from nearby cells. In conclusion, the damage pattern of CEES droplets is quite different from that of dissolved CEES, and a low level of phospholipid and triglyceride is beneficial for preventing droplets formation in preparing CEES solution.

A Mouse Model of Acute and Delayed Complications of Sulfur Mustard Analogue, 2-Chloroethyl Ethyl Sulfide

Background: Numerous studies have been conducted on humans, animals, and cell cultures exposed to Sulfur Mustard (SM). However, the precise mechanism and cause or long-term pattern of SM injuries are not well defined. There is no protocol available for treating people with severe eye, lung, and skin ailments. The current study aimed to develop an animal model of the acute and delayed complications of SM exposure.Materials and Methods: Two strains of mice (BALB/c and C57BL/6), 6-8 weeks old at the onset of the study, were exposed to 2-Chloroethyl Ethyl Sulfide (CEES) (1-200 mg/kg) and solvents (Tyrode’s solution, Polyethylene Glycol 300, herbal oil) for a duration of 12 hours to 7 months. The administration route was Intraperitoneal (IP) injection. The mortality rate, signs, and apparent side effects were explored. At the end of the study, the mice’s lung, liver, and spleen tissues were extracted and fixed for future histopathological assessments.Results: Tyrode’s solution and Polyethylene Glycol 300 solvents were not appropriate for the present research. Pathological features observed in BALB/c mice were better than the C57BL/6 mice. Overall, 10 mg/kg CEES was the most suitable dose, because it had the least mortality rate and demonstrated the most pathological findings, such as the infiltration of inflammatory cells and proliferation of fibroblasts and fibrotic tissue in the lung. Five months after the CEES administration, the delayed phase complications were studied. Conclusion: The IP injection of 10 mg/kg CEES to BALB/c mice imitates short- and long-term complications of SM exposure in humans. This model is useful for preventing and treating SM exposure.

Design, Synthesis, and Characterization of Binaphthalene Precursors as Photoactivated DNA Interstrand Cross-Linkers

Most recently, alkylation via photogenerated carbocations has been identified as a novel mechanism for photoinduced DNA interstrand cross-link (ICL) formation by bifunctional aryl compounds. However, most compounds showed a low efficiency for DNA cross-linking. Here, we have developed a series of new 1,1'-binaphthalene analogues that efficiently form DNA ICLs upon 350 nm irradiation via generated 2-naphthalenylmethyl cations. The DNA cross-linking efficiency depends on the substituents at position 4 of the naphthalene moiety as well as the leaving groups. Compounds with NO2, Ph, H, Br, or OMe substituents led to 2-4 times higher DNA ICL yields than those with a boronate ester group. Compounds with trimethylammonium salt as a leaving group showed slightly better cross-linking efficiency than those with bromo as a leaving group. Some of these compounds showed a better cross-linking efficiency than that of traditional alkylating agents, such as nitrogen mustard analogues or quinone methide precursors. These highly efficient photoactivated carbocation precursors allow determination and characterization of the adducts formed between the photogenerated naphthalenyl cations and four natural nucleosides, indicating that the alkylation sites for these naphthalene analogues are dG, dA, and dC.