RESUMEN
Sulfur mustard (SM) is a highly toxic chemical warfare agent. Exposure to SM results in various pathologies including skin lesions with subsequent impaired wound healing. To date, there are no effective treatments available. Here we discover a SM-triggered pathomechanism involving miR-497-5p and its target survivin which contributes to keratinocyte dysfunction. Transcriptome analysis using RNA-seq in normal human epidermal keratinocytes (NHEK) revealed that SM evoked differential expression of 1896 mRNAs and 25 miRNAs with many of these RNAs known to be involved in keratinocyte function and wound healing. We demonstrated that keratinocyte differentiation and proliferation were efficiently regulated by miRNAs induced in skin cells after exposure to SM. The inhibition of miR-497-5p counteracted SM-induced premature differentiation and stimulated proliferation of NHEK. In addition, we showed that microneedle-mediated transdermal application of lipid-nanoparticles containing miR-497-5p inhibitor restored survivin biosynthesis and cellular functionality upon exposure to SM using human skin biopsies. Our findings expand the current understanding of SM-associated molecular toxicology in keratinocytes and highlight miR-497-5p as feasible clinical target for specific skin therapy in SM-exposed patients and beyond.
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Queratinocitos , MicroARNs , Gas Mostaza , Piel , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , MicroARNs/metabolismo , MicroARNs/genética , Gas Mostaza/toxicidad , Piel/efectos de los fármacos , Piel/patología , Piel/metabolismo , Proliferación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Survivin/metabolismo , Survivin/genética , Sustancias para la Guerra Química/toxicidadRESUMEN
Malodorants comprise notoriously smelling mercaptans and might be applied for crowd control. Because exposure to malodorants may lead to irritation of the respiratory system, choking, and coma, bioanalytical verification of poisoning might be required in a medical and forensic context. We herein present the detection and identification of novel biomarkers of exposure to ethyl mercaptan, n-butyl mercaptan, tert-butyl mercaptan, and iso-amyl mercaptan. These alkyl thiol compounds were found to form disulfide adducts in human serum albumin (HSA) in plasma in vitro with the only non-disulfide-bridged Cys34 residue and with other residues being part of the disulfide-bridged pattern in HSA. After proteinase K-catalyzed proteolysis, adducts of all mercaptans were detected simultaneously as the tripeptide Cys34*ProPhe and the dipeptides Cys369*Tyr, ValCys316*, and Cysx*Ala (x denominates either Positions 91, 200, 253, 361, and/or 448) by a sensitive micro-liquid chromatography-electrospray ionization tandem mass spectrometry (µLC-ESI MS/MS) method working in the scheduled multiple reaction monitoring (sMRM) mode. Time- and concentration-dependent adduct formations while exposure and proteolysis were investigated and the suitability of adducts as biomarkers of exposure was elaborated. Adducts at Cys34 showed the lowest limits of identification (LOIs, 6 nM to 1.2 µM mercaptan in plasma) and superior stability in plasma at 37°C. Therefore, Cys34*ProPhe appears as the most promising target to prove exposure to mercaptans at least in vitro.
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Desensitization of nicotinic acetylcholine receptors (nAChRs) can be induced by overstimulation with acetylcholine (ACh) caused by an insufficient degradation of ACh after poisoning with organophosphorus compounds (OPCs). Currently, there is no generally applicable treatment for OPC poisoning that directly targets the desensitized nAChR. The bispyridinium compound MB327, an allosteric modulator of nAChR, has been shown to act as a resensitizer of nAChRs, indicating that drugs binding directly to nAChRs can have beneficial effects after OPC poisoning. However, MB327 also acts as an inhibitor of nAChRs at higher concentrations and can thus not be used for OPC poisoning treatment. Consequently, novel, more potent resensitizers are required. To successfully design novel ligands, the knowledge of the binding site is of utmost importance. Recently, we performed in silico studies to identify a new potential binding site of MB327, MB327-PAM-1, for which a more affine ligand, UNC0646, has been described. In this work, we performed ligand-based screening approaches to identify novel analogs of UNC0646 to help further understand the structure-affinity relationship of this compound class. Furthermore, we used structure-based screenings and identified compounds representing four new chemotypes binding to MB327-PAM-1. One of these compounds, cycloguanil, is the active metabolite of the antimalaria drug proguanil and shows a higher affinity towards MB327-PAM-1 than MB327. Furthermore, cycloguanil can reestablish the muscle force in soman-inhibited rat muscles. These results can act as a starting point to develop more potent resensitizers of nAChR and to close the gap in the treatment after OPC poisoning.
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Receptores Nicotínicos , Animales , Ligandos , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/efectos de los fármacos , Sitios de Unión , Compuestos de Piridinio/farmacología , Compuestos de Piridinio/química , Ratas , Relación Estructura-Actividad , Masculino , Unión Proteica , Simulación del Acoplamiento Molecular , Soman , Antagonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/químicaRESUMEN
Malodorants are mixtures containing mercaptans, which trigger the flight instinct upon exposure and might thus be deployed in military and civilian defense scenarios. Exposure to mercaptans might lead to unconsciousness, thus representing a possible threat for health. Therefore, we developed and validated a bioanalytical procedure for the simultaneous detection and identification of corresponding biomarkers for the verification of exposure to mercaptans. Disulfide-adducts of ethyl mercaptan (SEt), n-butyl mercaptan (SnBu), tert-butyl mercaptan (StBu) and iso-amyl mercaptan (SiAm) with cysteine (Cys) residues in human serum albumin (HSA) were formed by in vitro incubation of human plasma. After pronase-catalyzed proteolysis, reaction products were identified as adducts of the single amino acid Cys and the dipeptide cysteine-proline (Cys34Pro) detected by a sensitive µLC-ESI MS/MS method working in the scheduled multiple reaction monitoring (sMRM) mode. Dose-response studies showed linearity for the yield of Cys34Pro-adducts in the range from 6 nM to 300 µM of mercaptans in plasma and limits of identification (LOI) were in the range from 60 nM to 6 µM. Cys34-adducts showed stability for at least 6 days in plasma (37 °C). The presented disulfide-biomarkers expand the spectrum for bioanalytical verification procedures and might be helpful to prove exposure to malodorants.
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Cisteína , Disulfuros , Albúmina Sérica Humana , Compuestos de Sulfhidrilo , Humanos , Cisteína/química , Cisteína/sangre , Albúmina Sérica Humana/química , Disulfuros/química , Compuestos de Sulfhidrilo/química , Espectrometría de Masas en Tándem/métodos , Odorantes/análisis , Biomarcadores/sangreRESUMEN
Poisoning with organophosphorus compounds, which can lead to a cholinergic crisis due to the inhibition of acetylcholinesterase and the subsequent accumulation of acetylcholine (ACh) in the synaptic cleft, is a serious problem for which treatment options are currently insufficient. Our approach to broadening the therapeutic spectrum is to use agents that interact directly with desensitized nicotinic acetylcholine receptors (nAChRs) in order to induce functional recovery after ACh overstimulation. Although MB327, one of the most prominent compounds investigated in this context, has already shown positive properties in terms of muscle force recovery, this compound is not suitable for use as a therapeutic agent due to its insufficient potency. By means of in silico studies based on our recently presented allosteric binding pocket at the nAChR, i.e. the MB327-PAM-1 binding site, three promising MB327 analogs with a 4-aminopyridinium ion partial structure (PTM0056, PTM0062, and PTM0063) were identified. In this study, we present the synthesis and biological evaluation of a series of new analogs of the aforementioned compounds with a 4-aminopyridinium ion partial structure (PTM0064-PTM0072), as well as hydroxy-substituted analogs of MB327 (PTMD90-0012 and PTMD90-0015) designed to substitute entropically unfavorable water clusters identified during molecular dynamics simulations. The compounds were characterized in terms of their binding affinity towards the aforementioned binding site by applying the UNC0642 MS Binding Assays and in terms of their muscle force reactivation in rat diaphragm myography. More potent compounds were identified compared to MB327, as some of them showed a higher affinity towards MB327-PAM-1 and also a higher recovery of neuromuscular transmission at lower compound concentrations. To improve the treatment of organophosphate poisoning, direct targeting of nAChRs with appropriate compounds is a key step, and this study is an important contribution to this research.
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Receptores Nicotínicos , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/efectos de los fármacos , Animales , Masculino , Agentes Nerviosos/toxicidad , Ratas Wistar , Ratas , Intoxicación por Organofosfatos/tratamiento farmacológico , Diafragma/efectos de los fármacos , Diafragma/metabolismo , Relación Estructura-Actividad , Compuestos de Piridinio/farmacología , Compuestos de Piridinio/síntesis química , Compuestos de Piridinio/química , Contracción Muscular/efectos de los fármacos , Unión Neuromuscular/efectos de los fármacos , Sitios de UniónRESUMEN
The use of radar technology for non-contact measurement of vital parameters is increasingly being examined in scientific studies. Based on a systematic literature search in the PubMed, German National Library, Austrian Library Network (Union Catalog), Swiss National Library and Common Library Network databases, the accuracy of heart rate and/or respiratory rate measurements by means of radar technology was analyzed. In 37% of the included studies on the measurement of the respiratory rate and in 48% of those on the measurement of the heart rate, the maximum deviation was 5%. For a tolerated deviation of 10%, the corresponding percentages were 85% and 87%, respectively. However, the quantitative comparability of the results available in the current literature is very limited due to a variety of variables. The elimination of the problem of confounding variables and the continuation of the tendency to focus on the algorithm applied will continue to constitute a central topic of radar-based vital parameter measurement. Promising fields of application of research can be found in particular in areas that require non-contact measurements. This includes infection events, emergency medicine, disaster situations and major catastrophic incidents.
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Frecuencia Cardíaca , Radar , Frecuencia Respiratoria , Humanos , Frecuencia Cardíaca/fisiología , Frecuencia Respiratoria/fisiología , Monitoreo Fisiológico/métodos , AlgoritmosRESUMEN
We herein present for the first time the phosphylated (*) tetrapeptide (TP)-adduct GlyGluSer198*Ala generated from butyrylcholinesterase (BChE) with proteinase K excellently suited for the verification of exposure to toxic organophosphorus nerve agents (OPNA). Verification requires bioanalytical methods mandatory for toxicological and legal reasons. OPNA react with BChE by phosphonylation of the active site serine residue (Ser198) forming one of the major target protein adducts for verification. After its enzymatic cleavage with pepsin, the nonapeptide (NP) PheGlyGluSer*AlaGlyAlaAlaSer is typically produced as biomarker. Usually OPNA occur as racemic mixtures of phosphonic acid derivatives with the stereocenter at the phosphorus atom, e.g. (±)-VX. Both enantiomers react with BChE, but the adducted NP does not allow their chromatographic distinction. In contrast, the herein introduced TP-adducts appeared as two peaks when using a stationary reversed phase (1.8 µm) in micro-liquid chromatography-electrospray ionisation tandem-mass spectrometry (µLC-ESI MS/MS) analysis. These two peaks represent diastereomers of the (+)- and (-)-OPNA adducted to the peptide that comprises chiral L-amino acids exclusively. Concentration- and time-dependent effects of adduct formation with (±)-VX and its pure enantiomers (+)- and (-)-VX as well as with (±)-cyclosarin (GF) were investigated in detail characterising enantioselective adduct formation, stability, ageing and spontaneous reactivation. The method was also successfully applied to samples from a real case of pesticide poisoning as well as to samples of biomedical proficiency tests provided by the Organisation for the Prohibition of Chemical Weapons.
Asunto(s)
Sustancias para la Guerra Química , Agentes Nerviosos , Compuestos Organotiofosforados , Butirilcolinesterasa/metabolismo , Espectrometría de Masas en Tándem/métodos , Compuestos Organotiofosforados/toxicidad , Compuestos Organofosforados/toxicidad , Agentes Nerviosos/toxicidad , Sustancias para la Guerra Química/toxicidad , Sustancias para la Guerra Química/químicaRESUMEN
Intoxications with organophosphorus compounds (OPCs) based chemical warfare agents and insecticides may result in a detrimental overstimulation of muscarinic and nicotinic acetylcholine receptors evolving into a cholinergic crisis leading to death due to respiratory failure. In the case of the nicotinic acetylcholine receptor (nAChR), overstimulation leads to a desensitization of the receptor, which cannot be pharmacologically treated so far. Still, compounds interacting with the MB327 binding site of the nAChR like the bispyridinium salt MB327 have been found to re-establish the functional activity of the desensitized receptor. Only recently, a series of quinazoline derivatives with UNC0642 as one of the most prominent representatives has been identified to address the MB327 binding site of the nAChR, as well. In this study, UNC0642 has been utilized as a reporter ligand to establish new Binding Assays for this target. These assays follow the concept of MS Binding Assays for which by assessing the amount of bound reporter ligand by mass spectrometry no radiolabeled material is required. According to the results of the performed MS Binding Assays comprising saturation and competition experiments it can be concluded, that UNC0642 used as a reporter ligand addresses the MB327 binding site of the Torpedo-nAChR. This is further supported by the outcome of ex vivo studies carried out with poisoned rat diaphragm muscles as well as by in silico studies predicting the binding mode of UNC0646, an analog of UNC0642 with the highest binding affinity, in the recently proposed binding site of MB327 (MB327-PAM-1). With UNC0642 addressing the MB327 binding site of the Torpedo-nAChR, this and related quinazoline derivatives represent a promising starting point for the development of novel ligands of the nAChR as antidotes for the treatment of intoxications with organophosphorus compounds. Further, the new MS Binding Assays are a potent alternative to established assays and of particular value, as they do not require the use of radiolabeled material and are based on a commercially available compound as reporter ligand, UNC0642, exhibiting one of the highest binding affinities for the MB327 binding site known so far.
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Compuestos de Piridinio , Receptores Nicotínicos , Ratas , Animales , Receptores Nicotínicos/metabolismo , Ligandos , Relación Estructura-Actividad , Sitios de Unión , Quinazolinas , Compuestos Organofosforados , Torpedo/metabolismoRESUMEN
Trauma and toxic substances are connected in several aspects. On the one hand, toxic substances can be the reason for traumatic injuries in the context of accidental or violent and criminal circumstances. Examples for the first scenario is the release of toxic gases, chemicals, and particles during house fires, and for the second scenario, the use of chemical or biological weapons in the context of terroristic activities. Toxic substances can cause or enhance severe, life-threatening trauma, as described in this review for various chemical warfare, by inducing a tissue trauma accompanied by break down of important barriers in the body, such as the blood-air or the blood-gut barriers. This in turn initiates a "vicious circle" as the contribution of inflammatory responses to the traumatic damage enhances the macro- and micro-barrier breakdown and often results in fatal outcome. The development of sophisticated methods for detection and identification of toxic substances as well as the special treatment of the intoxicated trauma patient is summarized in this review. Moreover, some highly toxic substances, such as the protein toxins from the pathogenic bacterium Clostridioides (C.) difficile, cause severe post-traumatic complications which significantly worsens the outcome of hospitalized patients, in particular in multiply injured trauma patients. Therefore, novel pharmacological options for the treatment of such patients are necessarily needed and one promising strategy might be the neutralization of the toxins that cause the disease. This review summarizes recent findings on the molecular and cellular mechanisms of toxic chemicals and bacterial toxins that contribute to barrier breakdown in the human body as wells pharmacological options for treatment, in particular in the context of intoxicated trauma patients. "trauma-toxicology" comprises concepts regrading basic research, development of novel pharmacological/therapeutic options and clinical aspects in the complex interplay and "vicious circle" of severe tissue trauma, barrier breakdown, pathogen and toxin exposure, tissue damage, and subsequent clinical complications.
RESUMEN
Cyclooxygenase-2 (COX-2) is a key regulator of inflammation. High constitutive COX-2 expression enhances survival and proliferation of cancer cells, and adversely impacts antitumor immunity. The expression of COX-2 is modulated by various signaling pathways. Recently, we identified the melastatin-like transient-receptor-potential-7 (TRPM7) channel-kinase as modulator of immune homeostasis. TRPM7 protein is essential for leukocyte proliferation and differentiation, and upregulated in several cancers. It comprises of a cation channel and an atypical α-kinase, linked to inflammatory cell signals and associated with hallmarks of tumor progression. A role in leukemia has not been established, and signaling pathways are yet to be deciphered. We show that inhibiting TRPM7 channel-kinase in chronic myeloid leukemia (CML) cells results in reduced constitutive COX-2 expression. By utilizing a CML-derived cell line, HAP1, harboring CRISPR/Cas9-mediated TRPM7 knockout, or a point mutation inactivating TRPM7 kinase, we could link this to reduced activation of AKT serine/threonine kinase and mothers against decapentaplegic homolog 2 (SMAD2). We identified AKT as a direct in vitro substrate of TRPM7 kinase. Pharmacologic blockade of TRPM7 in wildtype HAP1 cells confirmed the effect on COX-2 via altered AKT signaling. Addition of an AKT activator on TRPM7 kinase-dead cells reconstituted the wildtype phenotype. Inhibition of TRPM7 resulted in reduced phosphorylation of AKT and diminished COX-2 expression in peripheral blood mononuclear cells derived from CML patients, and reduced proliferation in patient-derived CD34+ cells. These results highlight a role of TRPM7 kinase in AKT-driven COX-2 expression and suggest a beneficial potential of TRPM7 blockade in COX-2-related inflammation and malignancy.
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Leucemia Mielógena Crónica BCR-ABL Positiva , Leucemia Mieloide , Canales Catiónicos TRPM , Humanos , Proteínas Proto-Oncogénicas c-akt/genética , Ciclooxigenasa 2/genética , Canales Catiónicos TRPM/genética , Leucocitos Mononucleares/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Inflamación , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
VX is a highly toxic organophosphorus nerve agent that reacts with a variety of endogenous proteins such as serum albumin under formation of adducts that can be targeted by analytical methods for biomedical verification of exposure. Albumin is phosphonylated by the ethyl methylphosphonic acid moiety (EMP) of VX at various tyrosine residues. Additionally, the released leaving group of VX, 2-(diisopropylamino)ethanethiol (DPAET), may react with cysteine residues in diverse proteins. We developed and validated a microbore liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry (µLC-ESI MS/HR MS) method enabling simultaneous detection of three albumin-derived biomarkers for the analysis of rat plasma. After pronase-catalyzed cleavage of rat plasma proteins single phosphonylated tyrosine residues (Tyr-EMP), the Cys34(-DPAET)Pro dipeptide as well as the rat-specific LeuProCys448(-DPAET) tripeptide were obtained. The time-dependent adduct formation in rat plasma was investigated in vitro and biomarker formation during proteolysis was optimized. Biomarkers were shown to be stable for a minimum of four freeze-and-thaw cycles and for at least 24 h in the autosampler at 15 °C thus making the adducts highly suited for bioanalysis. Cys34(-DPAET)Pro was superior compared to the other serum biomarkers considering the limit of identification and stability in plasma at 37 °C. For the first time, Cys34(-DPAET)Pro was detected in in vivo specimens showing a time-dependent concentration increase after subcutaneous exposure of rats underlining the benefit of the dipeptide disulfide biomarker for sensitive analysis.
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Agentes Nerviosos , Animales , Ratas , Agentes Nerviosos/toxicidad , Agentes Nerviosos/química , Albúmina Sérica Humana/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Compuestos Organofosforados , Dipéptidos , Biomarcadores , TirosinaRESUMEN
An important target in toxicology is the ion channel known as human transient receptor potential ankyrin 1 (hTRPA1). It is triggered by a variety of chemicals, including the alkylating chemical warfare agent sulfur mustard (SM). The activation potentials of structural analogs including O- and sesquimustard, nitrogen mustards (HN1, HN2, and HN3), and related chemotherapeutic drugs (bendamustine, cycylophosphamide, and ifosfamide) were examined in the current study. The aequorin assay was used to measure changes in intracellular calcium levels in human hTRPA1 overexpressing HEK293 cells. The XTT assay was used to determine cytotoxicity. The data presented here highlight that all investigated alkylating substances, with the exception of cyclophosphamide and ifosfamide, cause the activation of hTRPA1. Cytotoxicity and activation of hTRPA1 were found to be related. Compounds with high reactivity had higher cytotoxicity and vice versa. However, inhibiting hTRPA1 with the specific inhibitor AP18 could not reduce the cytotoxicity induced by alkylating agents. As a result, hTRPA1 does not play a significant role in the cytotoxicity of alkylating agents.
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Ifosfamida , Compuestos de Mostaza Nitrogenada , Humanos , Canal Catiónico TRPA1 , Células HEK293 , Alquilantes/toxicidad , NitrógenoRESUMEN
Transient receptor potential (TRP) channels are important in the sensing of pain and other stimuli. They may be triggered by electrophilic agonists after covalent modification of certain cysteine residues. Sulfur mustard (SM) is a banned chemical warfare agent and its reactivity is also based on an electrophilic intermediate. The activation of human TRP ankyrin 1 (hTRPA1) channels by SM has already been documented, however, the mechanism of action is not known in detail. The aim of this work was to purify hTRPA1 channel from overexpressing HEK293 cells for identification of SM-induced alkylation sites. To confirm hTRPA1 isolation, Western blot analysis was performed showing a characteristic double band at 125 kDa. Immunomagnetic separation was carried out using either an anti-His-tag or an anti-hTRPA1 antibody to isolate hTRPA1 from lysates of transfected HEK293 cells. The identity of the channel was confirmed by micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry. Following SM exposure, hTRPA1 channel modifications were found at Cys462 and Cys665, as well as at Asp339 and Glu341 described herein for the first time. Since Cys665 is a well-known target of hTRPA1 agonists and is involved in hTRPA1 activation, SM-induced modifications of cysteine, as well as aspartic acid and glutamic acid residues may play a role in hTRPA1 activation. Considering hTRPA1 as a target of other SM-related chemical warfare agents, analogous adducts may be predicted and identified applying the analytical approach described herein.
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Sustancias para la Guerra Química , Gas Mostaza , Humanos , Gas Mostaza/toxicidad , Gas Mostaza/química , Canal Catiónico TRPA1/genética , Células HEK293 , Cisteína , Sustancias para la Guerra Química/toxicidad , Sustancias para la Guerra Química/química , AlquilaciónRESUMEN
(-)-Englerin A (EA), a potential novel anti-cancer drug, is a potent selective activator of classical transient receptor potential 4 and 5 (TRPC4, TRPC5) channels. As TRPC4 channels are expressed and functional in the lung endothelium, possible side effects such as lung edema formation may arise during its administration. Well-established in vivo rodent models for toxicological testing, however, rapidly degrade this compound to its inactive derivative, englerin B. Therefore, we chose an ex vivo isolated perfused and ventilated murine lung (IPVML) model to detect edema formation due to toxicants, which also reduces the number of incriminating animal experiments required. To evaluate the sensitivity of the IPVML model, short-time (10 min) drops of the pH from 7.4 down to 4.0 were applied, which resulted in linear changes of tidal volumes, wet-to-dry weight ratios and incorporation of FITC-coupled dextran particles from the perfusate. As expected, biological activity of EA was preserved after perfusion in the IPVML model. Concentrations of 50-100 nM EA continuously perfused through the IPVML model did not change tidal volumes and lung weights significantly. Wet-to-dry weight ratios were increased after perfusion of 100 nM EA but permeation of FITC-coupled dextran particles from the perfusate to the lung tissues was not significantly different. Therefore, EA shows little or no significant acute pulmonary toxicity after application of doses expected to activate target ion channels and the IPVML is a sensitive powerful ex vivo model for evaluating acute lung toxicity in accordance with the 3R rules for animal experimentation.
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Antineoplásicos , Canales Catiónicos TRPC , Animales , Antineoplásicos/toxicidad , Dextranos/metabolismo , Edema , Fluoresceína-5-Isotiocianato , Pulmón/metabolismo , Ratones , Perfusión , Sesquiterpenos de Guayano , Canales Catiónicos TRPC/metabolismoRESUMEN
For the verification of exposure to the banned blister agent sulfur mustard (SM) and the better understanding of its pathophysiology, protein adducts formed with endogenous proteins represent an important field of toxicological research. SM and its analogue 2-chloroethyl ethyl sulfide (CEES) are well known to alkylate nucleophilic amino acid side chains, for example, free-thiol groups of cysteine residues. The specific two-dimensional thiol difference gel electrophoresis (2D-thiol-DIGE) technique making use of maleimide dyes allows the staining of free cysteine residues in proteins. As a consequence of alkylation by, for example, SM or CEES, this staining intensity is reduced. 2D-thiol-DIGE analysis of human plasma incubated with CEES and subsequent matrix-assisted laser desorption/ionization time-of-flight (tandem) mass-spectrometry, MALDI-TOF MS(/MS), revealed transthyretin (TTR) as a target of alkylating agents. TTR was extracted from SM-treated plasma by immunomagnetic separation (IMS) and analyzed after tryptic cleavage by microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HR MS). It was found that the Cys10 -residue of TTR present in the hexapeptide C(-HETE)PLMVK was alkylated by the hydroxyethylthioethyl (HETE)-moiety, which is characteristic for SM exposure. It was shown that alkylated TTR is stable in plasma in vitro at 37°C for at least 14 days. In addition, C(-HETE)PLMVK can be selectively detected, is stable in the autosampler over 24 h, and shows linearity in a broad concentration range from 15.63 µM to 2 mM SM in plasma in vitro. Accordingly, TTR might represent a complementary protein marker molecule for the verification of SM exposure.
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Sustancias para la Guerra Química/análisis , Gas Mostaza/análogos & derivados , Prealbúmina/metabolismo , Alquilación , Biomarcadores/metabolismo , Sustancias para la Guerra Química/envenenamiento , Cromatografía Liquida/métodos , Electroforesis/métodos , Humanos , Gas Mostaza/análisis , Gas Mostaza/envenenamiento , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Factores de TiempoRESUMEN
Creatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine triphosphate (ATP) and creatine. The highly reactive free cysteine residue in the active site of the enzyme (Cys283) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys283 is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on rabbit muscle CK (rmCK) activity was investigated with special focus on the alkylation of Cys283 and of reactive methionine (Met) residues. For investigation of SM-alkylated amino acids in rmCK, micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry measurements were performed using the Orbitrap technology. The treatment of rmCK with SM resulted in a decrease of enzyme activity. However, this decrease did only weakly correlate to the modification of Cys283 but was conclusive for the formation of Met70-HETE and Met179-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met70Ala, Met179Leu, and Met70Ala/Met179Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met70 and Met179 residues for CK activity.
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Sustancias para la Guerra Química/toxicidad , Forma MM de la Creatina-Quinasa/efectos de los fármacos , Metionina/metabolismo , Gas Mostaza/toxicidad , Alquilación/efectos de los fármacos , Animales , Cromatografía Liquida , Forma MM de la Creatina-Quinasa/metabolismo , Cisteína/metabolismo , Conejos , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Sulfur mustard (SM) is a chemical warfare agent which use is banned under international law and that has been used recently in Northern Iraq and Syria by the so-called Islamic State. SM induces the alkylation of endogenous proteins like albumin and hemoglobin thus forming covalent adducts that are targeted by bioanalytical methods for the verification of systemic poisoning. We herein report a novel biomarker, namely creatine kinase (CK) B-type, suitable as a local biomarker for SM exposure on the skin. Human and rat skin were proven to contain CK B-type by Western blot analysis. Following exposure to SM ex vivo, the CK-adduct was extracted from homogenates by immunomagnetic separation and proteolyzed afterwards. The cysteine residue Cys282 was found to be alkylated by the SM-specific hydroxyethylthioethyl (HETE)-moiety detected as the biomarker tetrapeptide TC(-HETE)PS. A selective and sensitive micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HRMS) method was developed to monitor local CK-adducts in an in vivo study with rats percutaneously exposed to SM. CK-adduct formation was compared to already established DNA- and systemic albumin biomarkers. CK- and DNA-adducts were successfully detected in biopsies of exposed rat skin as well as albumin-adducts in plasma. Relative biomarker concentrations make the CK-adduct highly appropriate as a local dermal biomarker. In summary, CK or rather Cys282 in CK B-type was identified as a new, additional dermal target of local SM exposures. To our knowledge, it is also the first time that HETE-albumin adducts, and HETE-DNA adducts were monitored simultaneously in an in vivo animal study.
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Sustancias para la Guerra Química/toxicidad , Creatina Quinasa/metabolismo , Gas Mostaza/toxicidad , Piel/efectos de los fármacos , Albúminas/metabolismo , Alquilación/efectos de los fármacos , Animales , Biomarcadores/metabolismo , Cromatografía Liquida , Cisteína/metabolismo , Aductos de ADN/metabolismo , Humanos , Masculino , Ratas , Ratas Wistar , Piel/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Wound healing is a complex process, and disturbance of even a single mechanism can result in chronic ulcers developing after exposure to the alkylating agent sulfur mustard (SM). A possible contributor may be SM-induced chronic senescent mesenchymal stem cells (MSCs), unable to fulfil their regenerative role, by persisting over long time periods and creating a proinflammatory microenvironment. Here we show that senescence induction in human bone marrow derived MSCs was time- and concentration-dependent, and chronic senescence could be verified 3 weeks after exposure to between 10 and 40 µM SM. Morphological changes, reduced clonogenic and migration potential, longer scratch closure times, differences in senescence, motility and DNA damage response associated genes as well as increased levels of proinflammatory cytokines were revealed. Selective removal of these cells by senolytic drugs, in which ABT-263 showed initial potential in vitro, opens the possibility for an innovative treatment strategy for chronic wounds, but also tumors and age-related diseases.
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Senescencia Celular/efectos de los fármacos , Quimiocinas/metabolismo , Citocinas/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Gas Mostaza/toxicidad , Cicatrización de Heridas/efectos de los fármacos , Alquilantes/toxicidad , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Sustancias para la Guerra Química/toxicidad , Quimiocinas/genética , Citocinas/genética , Humanos , Peróxido de Hidrógeno/toxicidad , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Piel/efectos de los fármacos , Piel/lesionesRESUMEN
Herein, we report a novel paper-based electrochemical sensor for on-site detection of sulphur mustards. This sensor was conceived combining office paper-based electrochemical sensor with choline oxidase enzyme to deliver a sustainable sensing tool. The mustard agent detection relies on the evaluation of inhibition degree of choline oxidase, which is reversibly inhibited by sulphur mustards, by measuring the enzymatic by-product H2O2 in chronoamperometric mode. A nanocomposite constituted of Prussian Blue nanoparticles and Carbon Black was used as working electrode modifier to improve the electroanalytical performances. This bioassay was successfully applied for the measurement of a sulphur mustard, Yprite, obtaining a detection limit in the millimolar range (LOD = 0.9 mM). The developed sensor, combined with a portable and easy-to-use instrumentation, can be applied for a fast and cost-effective detection of sulphur mustards.