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The chemical warfare agent sulfur mustard (SM) affects all cells in the epidermis including melanocytes which are responsible for melanin synthesis. After exposure to SM, pigment abnormalities like hypo- and hyperpigmentation can occur. The underlying molecular pathomechanisms of SM exposure on human melanogenesis have not been elucidated so far. In our study, we investigated the effect of SM on human melanocytes and melanogenesis. Normal human epidermal melanocytes (NHEM) were used as in vitro model and they were exposed to different concentrations of SM (4.5 μM-100 μM). Melanin production was analyzed by absorption measurements at 405 nm. In addition, quantitative real-time PCR (qPCR) and Western blot experiments were performed to determine the expression of essential melanogenesis-related proteins including tyrosinase (TYR), tyrosinase-related protein (TRP) 1 and 2 and microphthalmia transcription factor (MITF). Our findings demonstrated that exposure to low SM concentrations increased melanin synthesis accompanied with an increase in protein expression. In contrast, high SM concentrations led to decreased melanin content and a downregulation in expression of all investigated melanogenesis-associated proteins. We concluded that low SM concentrations may cause hyperpigmentation while high SM concentrations decreased melanin content which may explain hypopigmented skin areas in SM exposed patients., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Vesicants cause a multitude of cutaneous reactions like erythema, blisters and ulcerations. After exposure to sulfur mustard (SM) and related compounds, patients present dermal symptoms typically known for chemicals categorized as skin sensitizer (e.g. hypersensitivity and flare-up phenomena). However, although some case reports led to the assumption that SM and other alkylating compounds represent sensitizers, a comprehensive investigation of SM-triggered immunological responses has not been conducted so far. Based on a well-structured system of in chemico and in vitro test methods, the Organization for Economic Co-operation and Development (OECD) established procedures to categorize agents on their skin sensitizing abilities. In this study, the skin sensitizing potential of SM and three related alkylating agents (AAs) was assessed following the OECD test guidelines. Besides SM, investigated AAs were chlorambucil (CHL), nitrogen mustard (HN3) and 2-chloroethyl ethyl sulfide (CEES). The methods are described in detail in the EURL ECVAM DataBase service on ALternative Methods to animal experimentation (DB-ALM). In accordance to OECD recommendations, skin sensitization is a pathophysiological process starting with a molecular initiating step and ending with the in vivo outcome of an allergic contact dermatitis. This concept is called adverse outcome pathway (AOP). An AOP links an adverse outcome to various key events which can be assayed by established in chemico and in vitro test methods. Positive outcome in two out of three key events indicates that the chemical can be categorized as a skin sensitizer. In this study, key event 1 "haptenation" (covalent modification of epidermal proteins), key event 2 "activation of epidermal keratinocytes" and key event 3 "activation of dendritic cells" were investigated. Covalent modification of epidermal proteins measured by using the DPRA-assay provided distinct positive results for all tested substances. Same outcome was seen in the KeratinoSens assay, investigating the activation of epidermal keratinocytes. The h-CLAT assay performed to determine the activation of dendritic cells provided positive results for SM and CEES but not for CHL and HN3. Altogether, following OECD requirements, our results suggest the classification of all investigated substances as skin sensitizers. Finally, a tentative AOP for SM-induced skin sensitization is suggested., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Sulfur mustard (SM) is a highly toxic chemical warfare agent, which produces blisters after skin contact. Treatment of SM-induced adverse health effects, such as cutaneous blistering, ulceration, and inflammation remains a challenging task. Antidotes or specific therapeutic measures are lacking. Some drugs (e.g. cyclooxygenase (COX) inhibitors) exhibited beneficial effects after SM poisoning in vivo. However, in vitro studies that evaluate and compare the potency of COX inhibitors are missing. In the presented study, non-specific (acetylsalicylic acid, ibuprofen, diclofenac, indomethacin, and piroxicam), COX-2-specific (celecoxib and parecoxib) inhibitors and COX-independent drugs (paracetamol and tofacitinib) were compared regarding anti-inflammatory and cytoprotective effects after SM exposure in post-exposure treatment settings. Normal human epidermal keratinocytes (NHEK) were used as a surrogate model. Prostaglandin E 2 (PGE 2 ) formation, a direct indicator for COX activity, was determined by ELISA. Changes in pro-inflammatory cytokine levels after SM exposures were assessed by quantitative determination of 27 inflammatory cytokines using a multiplex method. Cytotoxicity was determined using an XTT viability assay. The results demonstrated that SM highly increased PGE 2 production and release of pro-inflammatory cytokines, predominantly IL-6, IL-8 and TNF-α. In general, all COX inhibitors and paracetamol were able to reduce the PGE 2 formation, while tofacitinib, an inhibitor of Janus kinase, had no influence on PGE 2 levels. In addition, IL-6, IL-8, and TNF-α formation were also inhibited, but sometimes independently of PGE 2 . The COX-2 specific celecoxib was identified as the most potent drug to reduce IL-6, IL-8 and TNF-α formation after SM exposures in vitro. However, cell viability was not improved significantly by any of the investigated drugs in our experiments., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Although its first military use in Ypres was 100 years ago, no causal therapy for sulfur mustard (SM) intoxications exists so far. To improve the therapeutic options for the treatment of SM intoxications, we developed a co-culture of keratinocytes (HaCaT cells) and immunocompetent cells (THP-1 cells) to identify potential substances for further research. Here, we report on the influence of necrosulfonamide (NSA) on the course of a SM intoxication in vitro. The cells were challenged with 100, 200 and 300 μM SM and after 1 h treated with NSA (1, 5, 10 μM). NSA was chosen for its known ability to inhibit necroptosis, a specialized pathway of programmed necrosis. However, in our settings NSA showed only mild effects on necrotic cell death after SM intoxication, whereas it had an immense ability to prevent apoptosis. Furthermore, NSA was able to reduce the production of interleukin-6 and interleukin-8 at certain concentrations. Our data highlight NSA as a candidate compound to address cell death and inflammation in SM exposure., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Inhalation of the chemical warfare agent sulfur mustard (SM) is associated with severe acute and long-term pulmonary dysfunctions and health effects. The still not completely elucidated molecular toxicology and a missing targeted therapy emphasize the need for further research. However, appropriate human data are extremely rare. In vivo animal experiments are often regarded as gold standard in toxicology but may exhibit significant differences compared to the human pulmonary anatomy and physiology. Thus, alternative in vitro exposure methods, adapted to the human in vivo situation by exposing cells at the air-liquid interface (ALI), are complimentary approaches at a cellular level. So far, it is unclear whether the enhanced experimental complexity of ALI exposure, that is potentially biologically more meaningful, is superior to submerged exposures which are typically performed. Aim of our study was the evaluation of an appropriate in vitro exposure system (CULTEX ® Radial Flow System (RFS) equipped with an eFlow ® membrane nebulizer) for the exposure of cultivated human lung cells (A549) with SM under ALI conditions. Cellular responses (i.e. cell viability) and formation of SM-specific DNA-adducts were investigated and compared between ALI and submerse SM exposures. Our results proved the safe applicability of our ALI exposure system setup. The aerosol generation and subsequent deposition at the ALI were stable and uniform. The technical CULTEX ® RFS setup is based on ALI exposure with excess of aerosol from that only some is deposited on the cell layer. As expected, a lower cytotoxicity and DNA-adduct formation were detected when identical SM concentrations were used compared to experiments under submerged conditions. A distinct advantage of SM-ALI compared to SM-submerse exposures could not be found in our experiments. Though, the CULTEX ® RFS was found suitable for SM-ALI exposures., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Sulfur mustard (SM) is a vesicant agent who had its first military use 100 years ago, in Ypres. Since then it has been used in several conflicts like the Iran-Iraq war in the 1980s. The use of SM in Syria 2015 indicated the still existing threat. Despite decades of research no causal antidote against SM intoxication is available, so far. A SM intoxication is accompanied by necrosis, apoptosis and inflammation. To counteract the SM-induced inflammation, glucocorticoids and non-steroidal anti-inflammatory compounds (NSAIDs) are recommended. Aim of this study was to evaluate the efficacy of the anti-inflammatory compounds dexamethasone, ibuprofen and diclofenac in vitro. For that purpose, two different cell culture models were used. Firstly, a monoculture of keratinocytes (HaCaT) and secondly, an established co-culture of keratinocytes (HaCaT) and immunocompetent cells (THP-1) to identify the role of immune cells in the process and to mimic the dermal physiology more closely. Both models were challenged with different SM concentrations (100, 200 and 300μM) and treated with different anti-inflammatory compounds one hour after the SM exposure. Analytical analysis of necrosis (ToxiLight), apoptosis (CDDE) and inflammation (IL-6 and -8 ELISAs) followed 24h thereafter. Dexamethasone provided small but consistent protective effects in the monoculture. For the reduction of apoptosis, 3μM dexamethasone was sufficient. The most effective reduction regarding interleukin (IL) production was found with 6μM dexamethasone. Protective effects were less pronounced in co-culture, which implies, that the protective effects of dexamethasone are rather generic and not due to a modulation of the immune cells. Against our expectations, ibuprofen strongly amplified apoptosis and necrosis in SM exposed cells in the monoculture as well as the co-culture. Therefore, use of ibuprofen for treatment of SM intoxication should at least be considered most critically, if not even regarded as harmful. Diclofenac significantly reduced necrosis, apoptosis and inflammation in the co-culture in a dose-dependent manner. The greatest benefit regarding cell survival and reduction of the inflammation-marker IL-6 after a SM treatment was observed after diclofenac treatment. The protective effects of diclofenac were less pronounced in the monoculture which suggests, that diclofenac can modify the response of immune cells to SM. In conclusion, the results of our experiments, showing a benefit for diclofenac after SM exposure are in line with in vivo data of other researchers. Though, our in vitro results suggest the preferred use of diclofenac over ibuprofen. The benefit of dexamethasone is still equivocal, but low concentrations seem to have some positive effects., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Skin affections after sulfur mustard (SM) exposure include erythema, blister formation and severe inflammation. An antidote or specific therapy does not exist. Anti-inflammatory compounds as well as substances counteracting SM-induced cell death are under investigation. In this study, we investigated the benzylisoquinoline alkaloide berberine (BER), a metabolite in plants like berberis vulgaris, which is used as herbal pharmaceutical in Asian countries, against SM toxicity using a well-established in vitro approach. Keratinocyte (HaCaT) mono-cultures (MoC) or HaCaT/THP-1 co-cultures (CoC) were challenged with 100, 200 or 300mM SM for 1h. Post-exposure, both MoC and CoC were treated with 10, 30 or 50μM BER for 24h. At that time, supernatants were collected and analyzed both for interleukine (IL) 6 and 8 levels and for content of adenylate-kinase (AK) as surrogate marker for cell necrosis. Cells were lysed and nucleosome formation as marker for late apoptosis was assessed. In parallel, AK in cells was determined for normalization purposes. BER treatment did not influence necrosis, but significantly decreased apoptosis. Anti-inflammatory effects were moderate, but also significant, primarily in CoC. Overall, BER has protective effects against SM toxicity in vitro. Whether this holds true should be evaluated in future in vivo studies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The chemical warfare agent sulfur mustard (SM) can cause long-term health effects that may occur even years after a single exposure. The underlying pathophysiology is unknown, but epigenetic mechanisms are discussed as feasible explanation. "Epigenetics" depicts regulation of gene function without affecting the DNA sequence itself. DNA-methylation and covalent histone modifications (methylation or acetylation) are regarded as important processes. In the present in vitro study using early endothelial cells (EEC), we analyzed SM-induced DNA methylation over time and compared results to an in vivo skin sample that was obtained approx. one year after an accidental SM exposure. EEC were exposed to low SM concentrations (0.5 and 1.0μM). DNA methylation and histone acetylation (H3-K9, H3-K27, H4-K8) or histone di-methylation (H3-K9, H3-K27, H3-K36) were investigated 24h after exposure, and after 2 or 4 additional cell passages. The human skin sample was assessed in parallel. SM had only some minor effects on histone modifications. However, a significant and pronounced increase of DNA methylation was detected in the late cell passages as well as in the skin sample. Our results indicate that SM does indeed cause epigenetic modifications that appear to persist over time., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The hygroscopic zinc chloride (ZnCl 2 ) is often used to generate smoke screens. Severe adverse pulmonary health effects have been associated with inhalation of ZnCl 2 smokes. The underlying molecular toxicology is not known. Recent studies have shown that the Transient Receptor Potential Channel A1 (TRPA1) is important for sensing toxic chemicals. TRPA1 was shown to be activated by Zn 2+ which was linked to pain and inflammation. In the present study, we investigated whether TRPA1 activation contributes to ZnCl 2 -mediated toxicity in vitro. HEK wildtype (HEK-wt), TRPA1 overexpressing HEK (HEK-A1) and A549 lung cells, endogenously expressing TRPA1, were exposed to ZnCl 2 . Changes of intracellular calcium levels [Ca 2+ ] i and cell viability were assessed after ZnCl 2 exposure in all cell types, without or with TRPA1 inhibition. ZnCl 2 increased [Ca 2+ ] i through TRPA1 channels in a complex manner in both HEK-A1 and A549 cells while HEK-wt did not respond to ZnCl 2 . There was no difference in toxicity between HEK-wt and HEK-A1 cells after ZnCl 2 exposure. Inhibition of TRPA1 did not influence toxicity in all investigated cells. Thus, our in vitro results support the assumption that TRPA1 does not primarily mediate toxicity of ZnCl 2 and does probably not represent a therapeutic target to abate ZnCl 2 toxicity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Sulfur mustard (SM), a chemical warfare agent, causes DNA alkylation, which is believed to be the main cause of its toxicity. SM DNA adducts are commonly used to verify exposure to this vesicant. However, the required analytical state-of-the-art mass-spectrometry methods are complex, use delicate instruments, are not mobile, and require laboratory infrastructure that is most likely not available in conflict zones. Attempts have thus been made to develop rapid detection methods that can be used in the field. The analysis of SM DNA adducts (HETE-G) by immunodetection is a convenient and suitable method. For a diagnostic assessment, HETE-G levels must be determined in relation to the total DNA in the sample. Total DNA can be easily visualized by the use of fluorescent DNA dyes. This study examines whether SM and related compounds affect total DNA staining, an issue that has not been investigated before. After pure DNA was extracted from human keratinocytes (HaCaT cells), DNA was exposed to different S- and N-alkylating agents. Our experiments revealed a significant, dose-dependent decrease in the fluorescence signal of fluorescent dye-stained DNA after exposure to alkylating agents. After mass spectrometry and additional fluorescence measurements ruled out covalent modifications of ethidium bromide (EthBr) by SM, we assumed that DNA crosslinks caused DNA condensation and thereby impaired access of the fluorescent dyes to the DNA. DNA digestion by restriction enzymes restored fluorescence, a fact that strengthened our hypothesis. However, monofunctional agents, which are unable to crosslink DNA, also decreased the fluorescence signal. In subsequent experiments, we demonstrated that protons produced during DNA alkylation caused a pH decrease that was found responsible for the reduction in fluorescence. The use of an appropriate buffer system eliminated the adverse effect of alkylating agents on DNA staining with fluorescent dyes. An appropriate buffer system is thus crucial for DNA quantification with fluorescent dyes in the presence of alkylating compounds., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Sulfur mustard (SM) is a chemical warfare agent causing blistering, inflammation and ulceration of the skin. Thiol compounds such as glutathione (GSH) and N-acetylcysteine (NAC) have been suggested as potential antidotes. We investigated SM toxicity in a human keratinocyte cell line (HaCaT) and used GSH and NAC to counteract its cytotoxic effects. Cells were treated with 1, 5 or 10mM GSH or NAC and exposed to 30, 100 or 300μM SM. Different treatment regimens were applied to model extra- and intra-cellular GSH/NAC effects on SM toxicity. Necrosis, apoptosis and interleukin-6 and -8 levels were determined 24h post-exposure. Necrosis and apoptosis increased with SM dose. Interleukin-6 and -8 production peaked at 100μM and decreased at 300μM probably due to reduced ability for interleukin biosynthesis. Intracellular GSH/NAC diminished necrosis induced by 100μM SM. Extracellular GSH/NAC protected against necrosis and apoptosis induced by 100 and 300μM SM. Interleukin-6 and -8 production, induced by 100μM SM was reduced by GSH/NAC. However, low-dose GSH/NAC treatment of cells exposed to 300μM SM led to increased interleukin production. Thus, moderately poisoned cells are mostly responsible for SM-induced secretion of pro-inflammatory cytokines. GSH and NAC treatment can reduce SM-induced toxic effects. Protective effects were more pronounced by extracellular GSH or NAC administration. Rescue of severely poisoned cells may result in a strong secretion of pro- inflammatory cytokines. In summary, thiol compounds such as GSH or NAC constitute a promising approach to improve the therapy for SM injury. Additional intervention to prevent adverse effects of interleukin production might be beneficial., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Purpose: The purpose of this study was to dosimetrically compare 6- and 10-MV photon beam energies in high-risk prostate cancer patients of various body habitus using a volumetric modulated arc therapy (VMAT) radiation delivery technique. The objectives of the study were to evaluate whether dosimetric differences exist and to investigate whether differences are dependent on patient body habitus., Methods and Materials: Forty patients with various body habitus who had previously received treatment to the prostate and pelvic lymph nodes with VMAT techniques were chosen. Patients were planned in the Pinnacle(3) treatment planning system with double or triple SmartArc plans with 6- and 10-MV photon energies. All patients were optimized with the same planning objectives and normalized such that 95% of the planning target volume (PTV) received the prescription dose. Patients were evaluated for PTV and organ at risk (OAR) parameters for the bladder, rectum, small bowel, penile bulb, and sigmoid colon. Metrics used for comparison were D2%, D98%, homogeneity, conformity, and dose falloff for the PTV and D(2%), D(mean), V(80%), V(60%), and V(40%) for OARs. Statistical differences were evaluated with a paired-sample Wilcoxon signed rank test with a significance level of .05., Results: For the PTV, there were no statistically significant differences in D(mean), D(2cc), conformation number, and homogeneity index values, but the dose falloff parameters, R50 and R25, showed a median improvement of 6.7% (P<.01) and 6.2% (P<.01), respectively, with 10 MV. A correlation between patient anterior-posterior distance (d(AP)) and percentage reduction in R50 of 0.436% per centimeter (P<.01) was determined. For OARs, statistically significant reductions in dose metrics were found in the small bowel and bladder, but increases in the D(2cc) of 3.5% in the penile bulb (P<.01) and 0.2% in the rectum (P=.02) were shown with 10 MV. The use of 10 MV also demonstrated a statistically significant reduction in the total number of monitor units of 15.9% (P<.01) compared with 6 MV., Conclusions: The study showed that 10 MV provides a faster dose falloff than 6 MV for patients whose prostate and pelvic lymph nodes are treated using a VMAT technique irrespective of body habitus; however, the improvement in dose falloff is dependent on body habitus and increases as the patient body habitus increases., (Copyright © 2015 American Society for Radiation Oncology. Published by Elsevier Inc. All rights reserved.)

It is well established that a gradient of extracellular calcium within the epidermis regulates the differentiation of keratinocytes. However, the molecular mechanisms implicated in this process are not fully understood. RNA interference of the calcium-sensing receptor (CaSR) showed that CaSR is essential in calcium-induced differentiation of normal human epidermal keratinocytes (NHEKs) by increasing the levels of free intracellular calcium, which upregulates the expression of Wnt5a but not Wnt3a, Wnt4, and Dkk-1 in the cells. Subsequently, autocrine Wnt5a promotes the differentiation of NHEKs, determined by increased biosynthesis of keratin-1 and loricrin, whereas proliferation is suppressed. Addition of both Wnt5a and calcium to NHEKs activated the Wnt/β-catenin signaling pathway as indicated by (i) increased stability of β-catenin in the cells, (ii) enhanced β-catenin transcriptional activity, demonstrated by a luciferase-based β-catenin-activated reporter assay, and (iii) augmented Wnt/β-catenin target gene expression. NHEKs depleted for β-catenin had a significantly reduced susceptibility to calcium-induced differentiation. Knockdown of axin 2, an antagonist of β-catenin stability, enhanced the biosynthesis of keratin-1 and loricrin in the cells. Our findings establish a directional crosstalk between CaSR and Wnt/β-catenin signaling in keratinocyte differentiation via Wnt5a that acts as an autocrine stimulus in this process.

The chemical warfare agent sulfur mustard (SM) severely affects the regeneration capacity of skin. The underlying molecular and cellular mechanisms, however, are far from clear. Here, we demonstrate that normal human epidermal keratinocytes (NHEK) after exposure to SM strongly upregulated expression of keratin-1, involucrin, and loricrin, thus indicating premature epidermal differentiation. Furthermore, proliferation was repressed after treatment with SM. Analysis of intracellular signaling in NHEK revealed that SM enhances phosphorylation, nuclear translocation, and activity of the mitogen-activated protein kinases (MAPK) p38 and ERK1/2. Inhibition of p38 activity downregulated expression of keratin-1 and loricrin, whereas blockage of ERK1/2 significantly stimulated biosynthesis of these markers, pointing to opposite roles of p38 and ERK1/2 in the differentiation process. Simultaneous interruption of p38 and ERK1/2 activity led to a decreased expression of keratin-1 and loricrin. This suggests that NHEK differentiation is essentially controlled by p38 activity which may be negatively influenced by ERK1/2 activity. Functional analysis demonstrated that SM affects NHEK in their ability to migrate through extracellular matrix which can be rescued upon application of an inhibitor of p38 activity. Thus, our findings indicate that SM triggers premature differentiation in keratinocytes via p38 activity which may contribute to impaired regeneration of SM-injured skin., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Matrix metalloproteinases (MMPs), especially MMP-9 and MMP-2, degrade various proteins of the extracellular matrix, including collagen type IV the major component of basement membranes which also separate the epidermis from the dermis. Although previous work indicates the contribution of MMPs and their inhibitors (TIMPs) to the pathophysiology of skin lesions induced by the toxic chemical warefare agent sulphur mustard (SM), little is known about the underlying molecular and cellular mechanisms. In this study we demonstrate in a 3D-skin model that topical application of SM significantly upregulated basal MMP-9 mRNA expression and release from the cells as shown by qRT-PCR and zymography, whereas that of MMP-2, membrane-type 1 (MT1)-MMP, TIMP-1 and TIMP-2 remained almost unaffected by SM. Further studies in neonatal human dermal fibroblasts (NHDF) and HaCaT keratinocytes revealed that MMP-9 was not secreted from these cells, neither with or without exposure to SM. However, when NHDF and HaCaT were cocultivated, MMP-9 was expressed and released from the cell mixture, suggesting that interaction between both cell types is essential for MMP-9 production. Moreover, SM-treatment of NHDF/HaCaT cocultures further upregulated MMP-9 biosynthesis and secretion, which was consistent with our findings obtained in the 3D-skin model. Addition of conditioned medium derived from SM-exposed HaCaT cells to NHDF was able to stimulate MMP-9 secretion and also increased the migratory potential of NHDF as shown in a scratch-wound healing assay and a fluorescent cell invasion assay. In contrast, culture supernatants of SM-treated NHDF had not such an effect on HaCaT cells. Taken together, our findings provide first evidence that SM exposure of skin stimulates keratinocytes to release soluble factors which in turn induce enhanced MMP-9 secretion and invasiveness of fibroblasts in vitro. This provides a potential mechanism probably contributing to SM-evoked tissue injury in vivo.