Publications by authors named "Horst Thiermann"

217 Publications

Alkylated epidermal creatine kinase as a biomarker for sulfur mustard exposure: comparison to adducts of albumin and DNA in an in vivo rat study.

Arch Toxicol 2021 04 26;95(4):1323-1333. Epub 2021 Feb 26.

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937, Munich, Germany.

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 Cys 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 Cys 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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http://dx.doi.org/10.1007/s00204-021-03005-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8032612PMC
April 2021

Genomic Adaption and Mutational Patterns in a HaCaT Subline Resistant to Alkylating Agents and Ionizing Radiation.

Int J Mol Sci 2021 Jan 24;22(3). Epub 2021 Jan 24.

Bundeswehr Institute of Radiobiology Affiliated to the University of Ulm, Neuherbergstr. 11, D-80937 Munich, Germany.

Sulfur mustard (SM) is a chemical warfare agent that can damage DNA via alkylation and oxidative stress. Because of its genotoxicity, SM is cancerogenic and the progenitor of many chemotherapeutics. Previously, we developed an SM-resistant cell line via chronic exposure of the popular keratinocyte cell line HaCaT to increasing doses of SM over a period of 40 months. In this study, we compared the genomic landscape of the SM-resistant cell line HaCaT/SM to its sensitive parental line HaCaT in order to gain insights into genetic changes associated with continuous alkylation and oxidative stress. We established chromosome numbers by cytogenetics, analyzed DNA copy number changes by means of array Comparative Genomic Hybridization (array CGH), employed the genome-wide chromosome conformation capture technique Hi-C to detect chromosomal translocations, and derived mutational signatures by whole-genome sequencing. We observed that chronic SM exposure eliminated the initially prevailing hypotetraploid cell population in favor of a hyperdiploid one, which contrasts with previous observations that link polyploidization to increased tolerance and adaptability toward genotoxic stress. Furthermore, we observed an accumulation of chromosomal translocations, frequently flanked by DNA copy number changes, which indicates a high rate of DNA double-strand breaks and their misrepair. HaCaT/SM-specific single-nucleotide variants showed enrichment of C > A and T > A transversions and a lower rate of deaminated cytosines in the CpG dinucleotide context. Given the frequent use of HaCaT in toxicology, this study provides a valuable data source with respect to the original genotype of HaCaT and the mutational signatures associated with chronic alkylation and oxidative stress.
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http://dx.doi.org/10.3390/ijms22031146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865644PMC
January 2021

Chronic senescent human mesenchymal stem cells as possible contributor to the wound healing disorder after exposure to the alkylating agent sulfur mustard.

Arch Toxicol 2021 02 25;95(2):727-747. Epub 2021 Jan 25.

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937, Munich, Germany.

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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http://dx.doi.org/10.1007/s00204-020-02946-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870771PMC
February 2021

Adduct of the blistering warfare agent sesquimustard with human serum albumin and its mass spectrometric identification for biomedical verification of exposure.

Anal Bioanal Chem 2020 Nov 9;412(28):7723-7737. Epub 2020 Sep 9.

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937, Munich, Germany.

Apart from the well-known sulfur mustard (SM), additional sulfur-containing blistering chemical warfare agents exist. Sesquimustard (Q) is one of them and five times more blistering than SM. It is a common impurity in mustard mixtures and regularly found in old munitions but can also be used in pure form. Compared to the extensive literature on SM, very little experimental data is available on Q and no protein biomarkers of exposure have been reported. We herein report for the first time the adduct of Q with the nucleophilic Cys residue of human serum albumin (HSA) formed in vitro and introduce two novel bioanalytical procedures for detection. After proteolysis of this HSA adduct catalyzed either by pronase or by proteinase K, two biomarkers were identified by high-resolution tandem mass spectrometry (MS/HR MS), namely a dipeptide and a tripeptide, both alkylated at their Cys residue, which we refer to as HETETE-CP and HETETE-CPF. HETETE represents the Q-derived thio-alkyl moiety bearing a terminal hydroxyl group: "hydroxyethylthioethylthioethyl." Targeting both peptide markers from plasma, a micro liquid chromatography-electrospray ionization tandem mass spectrometry method working in the selected reaction monitoring mode (μLC-ESI MS/MS SRM) was developed and validated as well suited for the verification of exposure to Q. Fulfilling the quality criteria defined by the Organisation for the Prohibition of Chemical Weapons, the novel methods enable the detection of exposure to Q alone or in mixtures with SM. We further report on the relative reactivity of Q compared to SM. Based on experiments making use of partially deuterated Q as the alkylating agent, we rule out a major role for six-membered ring sulfonium ions as relevant reactive species in the alkylation of Cys. Furthermore, the results of molecular dynamics simulations are indicative that the protein environment around Cys allows adduct formation with elongated but not bulky molecules such as Q, and identify important hydrogen bonding interactions and hydrophobic contacts. Graphical abstract.
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http://dx.doi.org/10.1007/s00216-020-02917-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7550388PMC
November 2020

Identification of creatine kinase and alpha-1 antitrypsin as protein targets of alkylation by sulfur mustard.

Drug Test Anal 2021 Feb 18;13(2):268-282. Epub 2020 Sep 18.

Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany.

Sulfur mustard (SM) is a toxic chemical warfare agent deployed in several conflicts within the last 100 years and still represents a threat in terroristic attacks and warfare. SM research focuses on understanding the pathophysiology of SM and identifying novel biomarkers of exposure. SM is known to alkylate nucleophilic moieties of endogenous proteins, for example, free thiol groups of cysteine residues. The two-dimensional-thiol-differences in gel electrophoresis (2D-thiol-DIGE) technique is an initial proteomics approach to detect proteins with free cysteine residues. These amino acids are selectively labeled with infrared-maleimide dyes visualized after GE. Cysteine residues derivatized by alkylating agents are no longer accessible for the maleimide-thiol coupling resulting in the loss of the fluorescent signal of the corresponding protein. To prove the applicability of 2D-thiol-DIGE, this technology was exemplarily applied to neat human serum albumin treated with SM, to lysates from human cell culture exposed to SM as well as to human plasma exposed to CEES (chloroethyl ethyl sulfide, an SM analogue). Exemplarily, the most prominent proteins modified by SM were identified by matrix-assisted laser desorption/ionization time-of-flight (tandem) mass spectrometry, MALDI-TOF MS(/MS), as creatine kinase (CK) from human cells and as alpha-1 antitrypsin (A1AT) from plasma samples. Peptides containing the residue Cys of CK and Cys of A1AT were unambiguously identified by micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS) as being alkylated by SM bearing the specific hydroxyethylthioethyl-(HETE)-moiety. Both peptides might represent potential biomarkers of SM exposure. This is the first report introducing these endogenous proteins as targets of SM alkylation.
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http://dx.doi.org/10.1002/dta.2916DOI Listing
February 2021

In Vitro Interaction of Organophosphono- and Organophosphorothioates with Human Acetylcholinesterase.

Molecules 2020 Jul 2;25(13). Epub 2020 Jul 2.

Bundeswehr Institute of Pharmacology and Toxicology, D-80937 Munich, Germany.

The implementation of the Chemical Weapons Convention (CWC) in 1997 was a milestone in the prohibition of chemical warfare agents (CWA). Yet, the repeated use of CWA underlines the ongoing threat to the population. Organophosphorus (OP) nerve agents still represent the most toxic CWA subgroup. Defensive research on nerve agents is mainly focused on the "classical five", namely tabun, sarin, soman, cyclosarin and VX, although Schedule 1 of the CWC covers an unforeseeable number of homologues. Likewise, an uncounted number of OP pesticides have been produced in previous decades. Our aim was to determine the in vitro inhibition kinetics of selected organophosphono- and organophosphorothioates with human AChE, as well as hydrolysis of the agents in human plasma and reactivation of inhibited AChE, in order to derive potential structure-activity relationships. The investigation of the interactions of selected OP compounds belonging to schedule 1 (V-agents) and schedule 2 (amiton) of the CWC with human AChE revealed distinct structural effects of the -alkyl, --alkyl and ,-dialkyl residues on the inhibitory potency of the agents. Irrespective of structural modifications, all tested V-agents presented as highly potent AChE inhibitors. The high stability of the tested agents in human plasma will most likely result in long-lasting poisoning in vivo, having relevant consequences for the treatment regimen. In conclusion, the results of this study emphasize the need to investigate the biological effects of nerve agent analogues in order to assess the efficacy of available medical countermeasures.
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http://dx.doi.org/10.3390/molecules25133029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7412149PMC
July 2020

Assessment of α-amanitin toxicity and effects of silibinin and penicillin in different in vitro models.

Toxicol In Vitro 2020 Sep 27;67:104921. Epub 2020 Jun 27.

Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilian-University Munich, Goethestraße 33, 80336 Munich, Germany; Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937 Munich, Germany.

Silibinin (Sil) is used as hepatoprotective drug and is approved for therapeutic use in amanitin poisoning. In our study we compared Sil-bis-succinate (Sil), a water-soluble drug approved for i.v.-administration, with Sil solved in ethanol (Sil), which is normally used in research. We challenged monocultures or 3D-microtissues consisting of HepG2 cells or primary hepatocytes with α-amanitin and treated with SIL, SIL, penicillin and combinations thereof. Cell viability and the integrity of the microtissues was monitored. Finally, the expression of the transporters OATP1B1 and B3 was analyzed by qRT-PCR. We demonstrated that primary hepatocytes were more sensitive to α-amanitin compared to HepG2. Primary hepatocytes cultures were protected by Sil and Sil independent of penicillin from the cytotoxic effects of α-amanitin. Subsequent studies of the expression profile of the transporters OATP1B1/B3 revealed that primary hepatocytes do express both whereas in HepG2 cells they were hardly detectable. Our study showed that Sil has significant advantage over Sil with no additional benefit of penicillin. Moreover, HepG2 cells may not represent an appropriate model to investigate Amanita phalloides poisoning in vitro with focus on OATP transporters since these cells are lacking sensitivity towards α-amanitin probably due to missing cytotoxicity-associated transporters suggesting that primary hepatocytes should be preferred in this context.
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http://dx.doi.org/10.1016/j.tiv.2020.104921DOI Listing
September 2020

Organophosphorus compounds and oximes: a critical review.

Arch Toxicol 2020 07 6;94(7):2275-2292. Epub 2020 Jun 6.

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937, Munich, Germany.

Organophosphorus (OP) pesticides and nerve agents still pose a threat to the population. Treatment of OP poisoning is an ongoing challenge and burden for medical services. Standard drug treatment consists of atropine and an oxime as reactivator of OP-inhibited acetylcholinesterase and is virtually unchanged since more than six decades. Established oximes, i.e. pralidoxime, obidoxime, TMB-4, HI-6 and MMB-4, are of insufficient effectiveness in some poisonings and often cover only a limited spectrum of the different nerve agents and pesticides. Moreover, the value of oximes in human OP pesticide poisoning is still disputed. Long-lasting research efforts resulted in the preparation of countless experimental oximes, and more recently non-oxime reactivators, intended to replace or supplement the established and licensed oximes. The progress of this development is slow and none of the novel compounds appears to be suitable for transfer into advanced development or into clinical use. This situation calls for a critical analysis of the value of oximes as mainstay of treatment as well as the potential and limitations of established and novel reactivators. Requirements for a straightforward identification of superior reactivators and their development to licensed drugs need to be addressed as well as options for interim solutions as a chance to improve the therapy of OP poisoning in a foreseeable time frame.
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http://dx.doi.org/10.1007/s00204-020-02797-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367912PMC
July 2020

A novel high-performance liquid chromatography with diode array detector method for the simultaneous quantification of the enzyme-reactivating oximes obidoxime, pralidoxime, and HI-6 in human plasma.

Drug Test Anal 2020 Jul 23;12(7):938-947. Epub 2020 Apr 23.

Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany.

Oximes such as pralidoxime (2-PAM), obidoxime (Obi), and HI-6 are the only currently available therapeutic agents to reactivate inhibited acetylcholinesterase (AChE) in case of intoxications with organophosphorus (OP) compounds. However, each oxime has characteristic agent-dependent reactivating efficacy, and therefore the combined administration of complementary oximes might be a promising approach to improve therapy. Accordingly, a new high-performance liquid chromatography method with diode-array detection (HPLC-DAD) was developed and validated allowing for simultaneous or single quantification of 2-PAM, Obi, and HI-6 in human plasma. Plasma was precipitated using 5% w/v aqueous zinc sulfate solution and subsequently acetonitrile yielding high recoveries of 94.2%-101.0%. An Atlantis T3 column (150 × 2.1mm I.D., 3 μm) was used for chromatographic separation with a total run time of 15 min. Quantification was possible without interferences within a linear range from 0.12 to 120 μg/mL for all oximes. Excellent intra-day (accuracy 91.7%-98.6%, precision 0.5%-4.4%) and inter-day characteristics (accuracy 89.4%-97.4%, precision 0.4%-2.2%) as well as good ruggedness were found. Oximes in processed samples were stable for at least 12 h in the autosampler at 15°C as well as in human plasma for at least four freeze-thaw cycles. Finally, the method was applied to plasma samples of a clinical case of pesticide poisoning.
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http://dx.doi.org/10.1002/dta.2800DOI Listing
July 2020

Diagnostics and treatment of nerve agent poisoning-current status and future developments.

Ann N Y Acad Sci 2020 11 21;1479(1):13-28. Epub 2020 Mar 21.

Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany.

Although 193 states have committed to the Chemical Weapons Convention and 98% of the declared chemical weapons stockpiles have been destroyed so far, nerve agent poisoning remains a lingering threat. The recent dissemination of sarin in Syria, the assassination of Kim Jong-Nam in Malaysia, and the assault on Sergei Skripal in the United Kingdom underline the need for effective treatment. The current therapeutic options of a muscarinic receptor antagonist, an oxime, and an anticonvulsant have been unchanged for decades. Therefore, new therapeutic strategies, for example, bioscavengers and receptor-active substances, are promising concepts that have to be examined for their benefits and limitations. In order to facilitate rapid diagnosis in challenging clinical situations, point-of-care diagnostics and detection are of importance. Therapeutic guidance concerning the duration and success of the current oxime therapy via determination of the cholinesterase status can contribute to an optimal use of resources. In summary, the challenges of current and future therapies for nerve agent poisoning and key diagnostic devices will be discussed.
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http://dx.doi.org/10.1111/nyas.14336DOI Listing
November 2020

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface.

J Vis Exp 2020 02 23(156). Epub 2020 Feb 23.

Bundeswehr Institute of Pharmacology and Toxicology; Walther Straub Institute of Pharmacology and Toxicology, University of Munich.

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules. Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment.
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http://dx.doi.org/10.3791/60572DOI Listing
February 2020

Early diagnosis of nerve agent exposure with a mobile test kit and implications for medical countermeasures: a trigger to react.

BMJ Mil Health 2020 Apr 20;166(2):99-102. Epub 2020 Feb 20.

Military Medical Academy, National Poison Control Center, Belgrade, Serbia.

Recent uses of nerve agents underline the need of early diagnosis as trigger to react (initiating medical countermeasures, avoiding cross-contamination). As organophosphorus (OP) pesticide poisoning exerts the same pathomechanism, that is, inhibition of the pivotal enzyme acetylcholinesterase (AChE), a portable cholinesterase (ChE) test kit was applied in an emergency room for rapid diagnosis of OP poisoning. OP nerve agents or pesticides result in the inhibition of AChE. As AChE is also expressed on erythrocytes, patient samples are easily available. However, in most clinics only determination of plasma butyrylcholinesterase (BChE) is established which lacks a pathophysiological correlate, shows higher variability in the population and behaves different regarding inhibition by OP and reactivation by oximes. The ChE test kit helped to diagnose atypical cases of OP poisoning, for example, missing of typical muscarinic symptoms, and resulted in administration of pralidoxime, the oxime used in Serbia. The ChE test kit also allows an initial assessment whether an oxime therapy is successful. In one case report, AChE activity increased after oxime administration indicating therapeutic success whereas BChE activity did not. With only BChE at hand, this therapeutic effect would have been missed. As inhibition of AChE or BChE activity is determined, the CE-certified device is a global diagnostic tool for all ChE inhibitors including carbamates which might also be misused as chemical weapon. The ChE test kit is a helpful point-of-care device for the diagnosis of ChE inhibitor poisoning. Its small size and easy menu-driven use advocate procurement where nerve agent and OP pesticide exposure are possible.
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http://dx.doi.org/10.1136/jramc-2019-001310DOI Listing
April 2020

Impact of soman and acetylcholine on the effects of propofol in cultured cortical networks.

Toxicol Lett 2020 Apr 16;322:98-103. Epub 2020 Jan 16.

Experimental Anesthesiology Section, Department of Anesthesiology and Intensive Care Medicine, Eberhard-Karls-University, Waldhoernlestrasse 22, 72072, Tuebingen, Germany. Electronic address:

Patients intoxicated with organophosphorous compounds may need general anaesthesia to enable mechanical ventilation or for control of epileptiform seizures. It is well known that cholinergic overstimulation attenuates the efficacy of general anaesthetics to reduce spontaneous network activity in the cortex. However, it is not clear how propofol, the most frequently used intravenous anaesthetic today, is affected. Here, we investigated the effects of cholinergic overstimulation induced by soman and acetylcholine on the ability of propofol to depress spontaneous action potential activity in organotypic cortical slices measured by extracellular voltage recordings. Cholinergic overstimulation by co-application of soman and acetylcholine (10 μM each) did not reduce the relative inhibition of propofol (1.0 μM; mean normalized action potential firing rate 0.49 ± 0.06 of control condition, p < 0.001, Wilcoxon signed rank test) but clearly reduced its efficacy. Co-application of atropine (10 nM) did not improve the efficacy. Propofol preserved its relative inhibitory potential but did not produce a degree of neuronal depression which can be expected to assure hypnosis in humans. Since a combination with atropine did not improve its efficacy, an increase in dosage will probably be necessary when propofol is used in victims suffering from organophosphorous intoxication.
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http://dx.doi.org/10.1016/j.toxlet.2020.01.012DOI Listing
April 2020

Comparison of the toxicity of sulfur mustard and its oxidation products in vitro.

Toxicol Lett 2020 Mar 18;321:69-72. Epub 2019 Dec 18.

Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany; Walther-Straub-Institute of Pharmacology and Toxicology, LMU Munich, Germany.

The molecular toxicology of the chemical warfare agent sulfur mustard (SM) is still not completely understood. It has been suggested that in addition to SM itself also biotransformation products thereof mediate cytotoxicity. In the current study, we assessed this aspect by exposing a human hepatocyte cell line (HepG2) to SM or to its oxidation products sulfur mustard sulfoxide (SMO), sulfur mustard sulfone (SMO), and divinyl sulfone (DVS). Cytotoxicity, determined with the XTT assay, revealed a significant higher toxicity of SMO and DVS compared to SM while SMO had no effect at any concentration. The exact biotransformation of SM leading to SMO, SMO and finally DVS is unknown so far. Involvement of the CYP450 system is discussed and was also investigated in the presented study. Modulation of CYP1A2 activity, taken as a model enzyme for CYP450, affected cytotoxicity of SM, SMO or DVS significantly. Induction of CYP1A2 with omeprazole led to decreased cytotoxicity for all compounds whereas inhibition with cimetidine resulted in an increased cytotoxicity for SM, but not for SMO and DVS. Our results indicate a distinctive role of the CYP450 system in SM poisoning. Future studies should address the metabolic conversion of SM in more detail. Our data may suggest the well-tolerated drug omeprazole as a potential co-treatment after contact to SM.
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http://dx.doi.org/10.1016/j.toxlet.2019.12.015DOI Listing
March 2020

Validation of automated pipetting systems for cell culture seeding, exposure and bio-analytical assays in sulfur mustard toxicology.

Toxicol Lett 2020 Mar 3;320:80-86. Epub 2019 Dec 3.

Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany; Universität der Bundeswehr München, Faculty of Human Sciences, Department for Sports Sciences, 85577 Neubiberg, Germany. Electronic address:

In vitro cell culture experiments are highly important techniques to accelerate drug discovery, conduct safety testing and reduce the need for animal studies. Therefore, automatization may help to enhance the technical precision, reduce external (including operator's) influence on the data and thus improve reliability. Prior to application in scientific studies, validation of automated systems is absolutely necessary. In this study we present the validation of two combined automated pipetting systems to conduct toxicity studies in HaCaT cells consisting of cell seeding, noxious agent exposure and several assays to assess cell survival, apoptosis and interleukin production. After initial validation of pipetting accuracy, we compared homogeneity after automated seeding to plates seeded by expert laboratory technicians. Moreover, automated dispensing of a potentially unstable noxious agent was analyzed in terms of speed and consistency. We found a 2 % technical imprecision for the cell survival assay and 4.5-6 % for the other assays, bioluminescent and ELISA techniques. Thus, we could demonstrate the excellent technical precision of our assays. In a final step, we found that intraday variations, though acceptable, were much larger than technical variations and had to assume an intraday biological variability between different wells of the same experimental group.
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http://dx.doi.org/10.1016/j.toxlet.2019.12.003DOI Listing
March 2020

Screening of chiral shift reagents suitable to generically separate the enantiomers of V-agents by P-NMR spectroscopy.

Toxicol Lett 2020 Mar 2;320:28-36. Epub 2019 Dec 2.

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937, Munich, Germany. Electronic address:

Fourteen amino acids protected at the N-terminal and at their side chains were screened for resolving the enantiomers of V-agents by NMR. While none of the shift reagents tested showed really effective separation in proton NMR, two of them (BOC-Gln(Xan)-OH, 16, and Z-Arg(Z)-OH), 21, with 16 superior to 21) were found suitable to separate the enantiomers of all V-agent homologues involved in the test by P-NMR. Molar ratios investigated were 1:0.5, 1:1, 1:1.5, 1:2, and 1:3 with the V-agent set to 1 throughout the experiments. All these ratios were more or less effective, but 1:3 was found to separate the V-agents the most reliable way. It is postulated that three chiral solvating molecules are then coordinated around the organophosphate: ion pair formation with the amino nitrogen of the V agent side chain, hydrogen bonding provided by the PO unit, and extension of coordination at the phosphorus atom itself. After chiral separation of VX by semi-preparative LC-MS the enantiomers were examined with both configurations of 16 releasing four different P NMR peaks which correspond to four different complexes: R-S, R-R, S-R, and S-S. Comparing these results with literature data it is assumed that (+)-VX corresponds to the R configuration and (-)-VX to the S-configuration.
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http://dx.doi.org/10.1016/j.toxlet.2019.12.004DOI Listing
March 2020

Effect of sulfur mustard on melanogenesis in vitro.

Toxicol Lett 2020 Feb 27;319:197-203. Epub 2019 Nov 27.

Walther-Straub-Institute of Pharmacology and Toxicology, LMU Munich, 80336, Munich, Germany; Bundeswehr Institute of Radiobiology Affiliated to the University of Ulm, 80937, Munich, Germany.

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.
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http://dx.doi.org/10.1016/j.toxlet.2019.11.014DOI Listing
February 2020

[Chemical warfare agent poisoning].

Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 2019 Nov;62(11):1370-1377

Institut für Pharmakologie und Toxikologie der Bundeswehr, Neuherbergstr. 11, 80937, München, Deutschland.

Despite long-lasting international efforts to ban and disarm chemical warfare agents (CWAs), they pose an ongoing threat to the population. The reasons for this are existing remainders, inappropriately disposed of chemical munitions and availability of instructions for synthesis in open literature. Dissemination of CWAs during war, warlike conflicts and terrorist incidents has recently resulted in thousands of deaths. In this manuscript CWAs and comparable substances are presented and the signs and symptoms of poisoning with these substances are described. Aside from clear recommendations for the treatment of poisoning by the single groups of CWAs, parallels to well-known related poisonings including pathophysiological similarities are demonstrated. Moreover, aspects of detection, diagnosis and general management, such as decontamination, verification and antidote stockpiling, are described.According to the respective pathophysiological target, CWAs are classified as lung, skin, nerve and incapacitating agents. They are generally liquids at ambient room temperature and are more or less able to vaporise. In recent years, pharmaceutical-based agents (PBAs) came on board although they are not listed in the chemical warfare convention and therefore not listed as CWAs. Due to their high toxicity, however, they are mentioned here. PBAs comprise, for example, synthetic opioids which can act after inhalative respiration.During the rescue of affected victims, early detection of CWAs, restriction of access to the contaminated area and use of protective clothes and masks by first responders are necessary. Exposure should be terminated as soon as possible by removal of the victim from the hot zone and decontamination. The latter is also important to avoid secondary contamination of other persons or facilities located outside of the contaminated zone. According to the type of poisoning, therapy should be started as soon as possible.
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http://dx.doi.org/10.1007/s00103-019-03035-4DOI Listing
November 2019

Sulfur mustard alkylates steroid hormones and impacts hormone function in vitro.

Arch Toxicol 2019 11 12;93(11):3141-3152. Epub 2019 Sep 12.

Bundeswehr Institute of Pharmacology and Toxicology, 80937, Munich, Germany.

The chemical warfare agent sulfur mustard (SM) alkylates a multitude of biomacromolecules including DNA and proteins. Cysteine residues and nucleophilic nitrogen atoms in purine DNA bases are typical targets of SM but potentially every nucleophilic structure may be alkylated by SM. In the present study, we analyzed potential SM-induced alkylation of glucocorticoid (GC) hormones and functional consequences thereof. Hydrocortisone (HC), the synthetic betamethasone (BM) and dexamethasone (DEX) were chosen as representative GCs. Structural modifications were assessed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. The hypothesized alkylation was verified and structurally allocated to the OH-group of the C21 atom. The biological function of SM-alkylated GCs was investigated using GC-regulated dual-luciferase reporter gene assays and an ex vivo GC responsiveness assay coupled with real-time quantitative polymerase chain reaction (RT-qPCR). For the reporter gene assays, HEK293-cells were transiently transfected with a dual-luciferase reporter gene that is transcriptional regulated by a GC-response element. These cells were then incubated either with untreated or SM-derivatized HC, BM or DEX. Firefly-luciferase (Fluc) activity was determined 24 h after stimulation. Fluc-activity significantly decreased after stimulation with SM-pre-exposed GC dependent on the SM concentration. The ex vivo RT-qPCR-based assay for human peripheral leukocyte responsiveness to DEX revealed a transcriptional dysregulation of GC-regulated genes (FKBP5, IL1R2, and GILZ) after stimulation with SM-alkylated DEX. Our results present GCs as new biological targets of SM associated with a disturbance of hormone function.
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http://dx.doi.org/10.1007/s00204-019-02571-xDOI Listing
November 2019

Efficacy of an organophosphorus hydrolase enzyme (OpdA) in human serum and minipig models of organophosphorus insecticide poisoning.

Clin Toxicol (Phila) 2020 05 27;58(5):397-405. Epub 2019 Aug 27.

CSIRO Biocatalysis & Synthetic Biology Team, Black Mountain Science and Innovation Park, Canberra, Australia.

Current therapeutic options for organophosphorus (OP) insecticide self-poisoning including atropine and oximes are inadequate and case fatality may exceed 20%. An OP hydrolase enzyme, OpdA, has been used for environmental cleansing of OP insecticides and prevented death in rat and non-human primate models of OP insecticide poisoning if given very quickly after exposure. We here tested OpdA's ability to break down OP insecticides in human serum and in clinically relevant minipig models of OP insecticide poisoning. Human serum was spiked with seven diverse WHO Class II OP insecticides (chlorpyrifos, quinalphos, diazinon, dimethoate, fenthion, phenthoate, and profenofos) and the effect of OpdA on degradation measured. The pharmacodynamic and clinical effects of OpdA treatment were studied in Gottingen minipigs orally poisoned with agricultural formulations of dimethoate EC40 or methyl parathion EC60; pharmacodynamic effects were also assessed in profenofos EC50-poisoned pigs. OpdA effectively hydrolysed OP insecticides in human serum, with rates varying from 856 (SD 44) down to 0.107 (SD 0.01) moles of substrate hydrolysed/mole of enzyme/sec (k) for quinalphos and phenthoate, respectively, although at rates 2-3 log orders less than found in buffered solution. It showed clinical benefit in minipig models, reducing the dose of noradrenaline required to sustain an adequate mean arterial pressure after dimethoate (mean 0.149 [SD 0.10] μg/kg/h vs. 1.07 [SD 0.77] μg/kg/h,  < .0001) and methyl parathion (mean 0.077 [SD 0.08] μg/kg/h vs. 0.707 [SD 0.49] μg/kg/h,  < .0001) poisoning. OpdA reduced blood OP insecticide concentration and acetylcholinesterase inhibition after poisoning by dimethoate, methyl parathion, and profenofos insecticides. incubation of OpdA in human serum showed hydrolysis of diverse OP insecticides, although at lower rates than found in buffer solutions. This activity results in clinical and pharmacodynamic efficacy against several OP insecticides. These results support the testing of OpdA in further animal models before considering human trials to determine whether it may become an urgently required novel therapeutic agent for OP insecticide self-poisoning.
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http://dx.doi.org/10.1080/15563650.2019.1655149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7114914PMC
May 2020

Skin sensitizing effects of sulfur mustard and other alkylating agents in accordance to OECD guidelines.

Toxicol Lett 2019 Oct 9;314:172-180. Epub 2019 Aug 9.

Walther-Straub-Institute of Pharmacology and Toxicology, LMU Munich, 80336 Munich, Germany; Bundeswehr Medical Service Academy, 80937, Munich, Germany.

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.
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http://dx.doi.org/10.1016/j.toxlet.2019.07.023DOI Listing
October 2019

Forensic evidence of sulfur mustard exposure in real cases of human poisoning by detection of diverse albumin-derived protein adducts.

Arch Toxicol 2019 07 8;93(7):1881-1891. Epub 2019 May 8.

Bundeswehr Institute of Pharmacology and Toxicology (InstPharmToxBw), Neuherbergstr. 11, 80937, Munich, Germany.

We present the forensic analyses of plasma samples of human victims exposed to sulfur mustard (SM) in a crisis region in the Middle East in 2015. A few hours after exposure, poisoned persons showed typical signs and symptoms of percutaneous SM exposure including erythema and later on blisters and hardly healing skin wounds. Blood samples were collected 15 days after poisoning to be analyzed for the presence of long-lived protein-adduct biomarkers to verify SM poisoning. We applied a novel bioanalytical toolbox targeting four human serum albumin-derived biomarkers that were made accessible after plasma proteolysis. These adducts contained the SM-specific hydroxyethylthioethyl moiety either bound to the thiol group of a cysteine residue (C*) or to the side-chain carboxylic group of a glutamic acid residue (E*). Peptide biomarkers were produced from plasma of the victims using proteinase K (C*PF), pronase (C*P) and pepsin (AE*VSKL and LQQC*PFEDHVKL) for enzymatic protein cleavage. Separation and detection were carried out by selective micro-liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry (µLC-ESI MS/HR MS). In addition to this site-specific adduct detection, a general approach after alkaline hydrolysis of the plasma protein fraction was applied. Liberated thiodiglycol (TDG) was derivatized with heptafluorobutyric anhydride and detected by gas chromatography-electron ionization mass spectrometry (GC-EI MS). The different bioanalytical methods yielded congruent results confirming SM poisoning for all patients who showed clinical signs and symptoms. This is the first time that real cases of SM poisoning were confirmed and presented by such a broad compilation of protein-derived biomarkers.
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http://dx.doi.org/10.1007/s00204-019-02461-2DOI Listing
July 2019

Evaluation of selective and non-selective cyclooxygenase inhibitors on sulfur mustard-induced pro-inflammatory cytokine formation in normal human epidermal keratinocytes.

Toxicol Lett 2019 Sep 15;312:109-117. Epub 2019 May 15.

Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians-University Munich, Goethestr. 33, 80336 Munich, Germany; Bundeswehr Medical Academy, Division F, Medical CBRN Defense, Neuherbergstr. 11, 80937 Munich, Germany. Electronic address:

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 (PGE) 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 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 formation, while tofacitinib, an inhibitor of Janus kinase, had no influence on PGE levels. In addition, IL-6, IL-8, and TNF-α formation were also inhibited, but sometimes independently of PGE. 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.
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http://dx.doi.org/10.1016/j.toxlet.2019.03.012DOI Listing
September 2019

Validation of the CULTEX® Radial Flow System for the assessment of the acute inhalation toxicity of airborne particles.

Toxicol In Vitro 2019 Aug 16;58:245-255. Epub 2019 Mar 16.

Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany; Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany.

The CULTEX® Radial Flow System (RFS) is a modular in vitro system for the homogenous exposure of cells to airborne particles at the air-liquid interface (ALI). A former pre-validation study successfully demonstrated the general applicability of the CULTEX® RFS and its transferability, stability and reproducibility. Based on these results, the methodology was optimized, validated and prediction models for acute inhalation hazards were established. Cell viability of A549 cells after ALI exposure to 20 pre-selected test substances was assessed in three independent laboratories. Cytotoxicity of test substances was compared to the respective incubator controls and used as an indicator of toxicity. Substances were considered to exert an acute inhalation hazard when viability decreased below 50% (prediction model (PM) 50%) or 75% (PM 75%) at any of three exposure doses (25, 50 or 100 μg/cm). Results were then compared to existing in vivo data and revealed an overall concordance of 85%, with a specificity of 83% and a sensitivity of 88%. Depending on the applied PM, the within-laboratory and between-laboratory reproducibility ranged from 90 to 100%. In summary, the CULTEX® RFS was proven as a transferable, reproducible and well predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles.
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http://dx.doi.org/10.1016/j.tiv.2019.03.020DOI Listing
August 2019

The arrhythmogenic potential of nerve agents and a cardiac safety profile of antidotes - A proof-of-concept study using human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM).

Toxicol Lett 2019 Jun 8;308:1-6. Epub 2019 Mar 8.

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstr. 11, 80937 Munich, Germany. Electronic address:

The global use of organophosphorus compounds (OP) for pest control and nerve agents being used in military conflicts and for assassinations renders intoxications by these agents a public health concern. OP-poisoned patients often suffer from dysrhythmias which may ultimately result in death. In this study, human-induced pluripotent stem cells derived cardiomyocytes were exposed to OP compounds in a microelectrode array system (MEA). The MEA system is widely accepted to assess the proarrhythmic properties of (candidate) drugs. The directly acting cholinergic compounds acetylcholine and carbachol and the irreversible acetylcholinesterase inhibitor cyclosarin - a highly toxic nerve agent - were assessed. All three compounds induced a dose-dependent (up to 600 nmol/L) corrected field potential duration (FPDc) prolongation of 9.7 ± 0.6% for carbachol, for 9.7 ± 1.2% acetylcholine and 9.4 ± 0.5% for cyclosarin. Additionally, the electrophysiological alterations of the clinically approved oxime reactivators obidoxime, pralidoxime and the oximes in development HI-6 and MMB-4 were investigated in the absence of OP. Neither of these oximes (up to a concentration of 300 μmol/L) caused dysrhythmia nor beat arrest. The competitive muscarinic receptor antagonist atropine as a cornerstone in the treatment of OP poisoning was also analyzed. Interestingly, atropine caused a drop in the beat rate which might result from a non-receptor action of this substance in the absence of OP. Atropine in combination with the OP nerve agent cyclosarin and the direct cholinergics acetylcholine or carabachol completely reversed the induced FPDc prolongation. However, the oxime HI-6 as potent reactivator of cyclosarin-inhibited AChE was not able to prevent the FPDc prolongation in this model. In conclusion, the current model allows the assessment of FPDc prolongation by the nerve agent cyclosarin, the cholinergic compounds carbachol, acetylcholine and the block of this effect by atropine.
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http://dx.doi.org/10.1016/j.toxlet.2019.03.003DOI Listing
June 2019

Human small bowel as model for poisoning with organophosphorus compounds.

Toxicol In Vitro 2019 Jun 11;57:76-80. Epub 2019 Feb 11.

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstr. 11, 80937 Munich, Germany. Electronic address:

In previous experiments, human and rat small bowel samples have been successfully used to study the spasmolytic effect of (potential) therapeutics in carbamate-constricted bowel specimens. Additionally, transferability from rat to human data was shown in the previous study. In the present study, the effects of atropine, scopolamine, MB327, HI-6 as well as obidoxime were examined in organophosphorus-poisoned human small bowel specimens. All substances were tested with at least seven concentrations in samples previously exposed to the nerve agent sarin. Furthermore, the cholinesterase reactivation potential of all substances was investigated. The test substances displayed a spasmolytic effect allowing the calculation of dose-response curves and ECs. The parasympatholytic compound scopolamine had the strongest relaxing effect (EC = 0.05 μM) followed by atropine (EC = 0.07 μM). HI-6 and obidoxime were capable to reactivate the sarin-inhibited cholinesterase activity in small bowel samples. Both substances restored AChE activity in a dose-dependent way, with HI-6 being more potent (HI-6 EC = 3.8 μM vs obidoxime EC = 197.8 μM). Summarizing, our isolated human small bowel setup is a suitable tool to investigate the smooth muscle relaxing effect of (candidate) therapeutics for organophosphorus compound poisoning i.e. sarin exposure in a complex 3D tissue model.
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http://dx.doi.org/10.1016/j.tiv.2019.02.010DOI Listing
June 2019

COPD and asthma therapeutics for supportive treatment in organophosphate poisoning.

Clin Toxicol (Phila) 2019 07 30;57(7):644-651. Epub 2019 Jan 30.

a Bundeswehr Institute of Pharmacology and Toxicology , Neuherbergstraße 11, Munich , Germany.

Nerve agents like sarin or VX have repeatedly been used in military conflicts or homicidal attacks, as seen in Syria or Malaysia 2017. Together with pesticides, nerve agents assort as organophosphorus compounds (OP), which inhibit the enzyme acetylcholinesterase. To counteract subsequent fatal symptoms due to acetylcholine (ACh) accumulation, oximes plus atropine are administered, a regimen that lacks efficacy in several cases of OP poisoning. New therapeutics are in development, but still need evaluation before clinical employment. Supportive treatment with already approved drugs presents an alternative, whereby compounds from COPD and asthma therapy are likely options. A recent pilot study by Chowdhury et al. included β2-agonist salbutamol in the treatment of OP-pesticide poisoned patients, yielding ambiguous results concerning the addition. Here, we provide experimental data for further investigations regarding the value of these drugs in OP poisoning. By video-microscopy, changes in airway area were analyzed in VX-poisoned rat precision cut lung slices (PCLS) after ACh-induced airway contraction and subsequent application of selected anticholinergics/β2-agonists. Glycopyrrolate and ipratropium efficiently antagonized an ACh-induced airway contraction in VX-poisoned PCLS (EC glycopyrrolate 15.8 nmol/L, EC ipratropium 2.3 nmol/L). β2-agonists formoterol and salbutamol had only negligible effects when solely applied in the same setting. However, combination of formoterol or salbutamol with low dosed glycopyrrolate or atropine led to an additive effect compared to the sole application [50.6 ± 8.8% airway area increase after 10 nmol/L formoterol +1 nmol/L atropine versus 11.7 ± 9.2% (10 nmol/L formoterol) or 8.6 ± 5.9% (1 nmol/L atropine)]. We showed antagonizing effects of anticholinergics and β2-agonists on ACh-induced airway contractions in VX-poisoned PCLS, thus providing experimental data to support a prospective comprehensive clinical study. Our results indicate that COPD and asthma therapeutics could be a valuable addition to the treatment of OP poisoning.
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http://dx.doi.org/10.1080/15563650.2018.1540785DOI Listing
July 2019

A novel exposure system generating nebulized aerosol of sulfur mustard in comparison to the standard submerse exposure.

Chem Biol Interact 2019 Jan 28;298:121-128. Epub 2018 Nov 28.

Bundeswehr Institute of Pharmacology and Toxicology, 80937, Munich, Germany; Walther Straub Institute of Pharmacology and Toxicology, University of Munich, 80336, Munich, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cbi.2018.11.025DOI Listing
January 2019

Methionine in human serum albumin: A novel target for alkylation by sulfur mustard.

Drug Test Anal 2019 May 22;11(5):659-668. Epub 2019 Jan 22.

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937, Munich, Germany.

Exposure to the vesicant sulfur mustard (SM) may lead to erythema and blistering. Toxicity of SM is hypothesized due to the alkylation of DNA bases and nucleophilic amino acid side chains in proteins (adducts) by forming the hydroxyethylthioethyl (HETE) moiety. Despite its prohibition by the chemical weapons convention, SM still represents a serious threat to military personnel and civilians. Therefore, development and improvement of forensic analytical methods for the verification of SM exposure is of high interest. Protein adducts have been shown to be highly suitable and beneficial biomarkers of poisoning. Herein we present methionine in human serum albumin (HSA) as a novel target of SM forming a HETE-methionyl sulfonium ion. The alkylated tetrapeptide LeuGlyMet (-HETE)Phe, LGM(-HETE)F, was detected after pepsin-mediated proteolysis and subsequent analysis by microbore liquid chromatography-electrospray ionization-high-resolution tandem-mass spectrometry. Compound identity was confirmed by a synthetic reference. Proteolysis conditions for HSA were optimized towards maximum yield of LGM(-HETE)F and its limit of identification (32.3 nM SM in serum) was similar to those of the established HSA-derived biomarkers HETE-CysPro and HETE-CysProPhe (15.6 nM SM in serum). Stability of the alkylated Met in vitro and in vivo was limited to 5 days making this modification a beneficial short-time biomarker. Furthermore, it was found that the HETE-methionyl sulfonium ion can transfer its HETE moiety to the side chain of cysteine and glutamic acid as well as to the N-terminus of peptides and proteins in vitro thus revealing novel insights into the molecular toxicity of SM.
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http://dx.doi.org/10.1002/dta.2548DOI Listing
May 2019

Collision-induced mass spectrometric fragmentation of protonated dimethoate and omethoate generated by electrospray ionization.

Rapid Commun Mass Spectrom 2019 Feb;33(3):259-271

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937, Munich, Germany.

Rationale: Dimethoate (DIM, S=P(OMe) -S-CH -C(O)-NH-CH ) is a dimethyl phosphorodithioate pesticide widely used in agri- and horticulture that undergoes biotransformation in vivo by desulfuration into its more toxic oxono-derivative omethoate (OM, O=P(OMe) -S-CH -C(O)-NH-CH ). OM inhibits acetylcholinesterase thus provoking cholinergic crisis in vivo, ultimately leading to death. Quantitative approaches for the determination of DIM and OM in environmental and toxicological samples make use of tandem mass spectrometry (MS ). Nevertheless, so far interpretation of resulting product ions is incomplete and sometimes contradictory.

Methods: DIM and OM as well as their deuterated analogues (fully deuterated at both methoxy groups bound to the phosphorus atom) were analyzed by MS and MS after positive electrospray ionization and collision-induced dissociation (CID) in a linear ion trap to characterize fragmentations. The accurate masses of product ions were determined in a time-of-flight mass analyzer. Hydrogen/deuterium (H/D)-exchange experiments were carried out for further support of product ion identification. In addition, density functional theory (DFT) computations were used to calculate both the most stable protonation sites of DIM and OM and the changes in the diverse bond lengths after protonation.

Results: Some identical and some related product ions of DIM and OM were found but also striking individual differences. Fragmentation pathways were proposed and product ions identified. Most fragmentations followed the common rules of charge migration fragmentation. DFT calculations supported experimental findings.

Conclusions: Discrepancies present in the literature so far are clarified and a deeper insight is provided into the fragmentation processes of organophosphorus pesticides. The combination of diverse experimental and theoretical approaches yielded consistent results, thus demonstrating continuous progress in understanding gas-phase reactions in MS experiments.
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http://dx.doi.org/10.1002/rcm.8343DOI Listing
February 2019