18^th Medical Chemical Defense Conference (MCDC), Munich
Selected Abstracts

Session 1: Implications of Recent Use of Nerve Agents

T02 • Case Presentation – Novichok Nerve Agent Poisoning

David Steindl

Case presentation of a 44-year-old man who developed confusion, increased diaphoresis and emesis followed by unconsciousness on a flight in Russia. After 2 days of stabilisation and treatment in a local toxicology unit, the patient was transferred to Charité – Universitätsmedizin Berlin, Germany. Subsequently, he was diagnosed with severe cholinesterase inhibitor poisoning. The patient was successfully treated with prolonged mechanical ventilation, basic intensive care, and atropine. On day 33, the patient was discharged with minimal neurologic findings, which had entirely resolved until follow-up on day 55. 2 weeks after admission, the Bundeswehr Institute of Pharmacology and Toxicology had identified an organophosphorus nerve agent from the novichok group in blood samples collected immediately after the patient’s admission to Charité, a finding that the Organisation for the Prohibition of Chemical Weapons subsequently confirmed.

Session 2: Advances in Medical Countermeasures over the Last Years

T08 • Chemical Warfare Agents – New Insights into the Molecular Toxicology

Dirk Steinritz

Since the first use of pulmonary agents and vesicants in World War I, as well as the development of nerve agents and their deployment in the first Gulf War, researchers have been studying the molecular toxicology of these compounds. Significant progress has been achieved during decades of research: specific molecular targets have been found, diagnostic tools and therapies have been developed and introduced. But antidotes are available only for specific chemical warfare agents, while for others they are completely unavailable. Furthermore, existing antidotes are sometimes ineffective, indicating that significant gaps remain.

This talk will discuss the most recent advances in medical chemical warfare agent research and will highlight issues and gaps that should be addressed on the molecular level in the future. Above all, optimal conditions (funding and political support) must exist to allow scientists to conduct the required experiments in an acceptable and cutting-edge manner. Moreover, a single institute is unlikely to provide complete technology and knowledge at the required level. Thus, research in this field should also take place through networks.

Session 3: Poster Award Competition

First place

P39 • Determination of Organophosphorus Compound Inhibited AChE Activity: Precision Cut Lung Slices (PCLS) as a Useful Model for AChE Reactivator Research

Fee Gölitz; Timo Wille; Franz Worek

Exposure to organophosphorus nerve agents (OPNA) leads to an acetylcholine overflow and disturbance of multiple organ functions, e.g., bronchoconstriction and bronchorrhea. Despite research for over more than 60 years, the efficacy of OPNA treatment is still limited and improved medical treatment protocols are needed. PCLS can be used as a relevant model to investigate possible new therapeutics. In this study, the AChE activity and the airway response were measured in intact PCLS after different treatments.

The colorimetric Ellman assay was modified to determine the AChE activity in intact PCLS. Here, Cyclosarin (GF), sarin (GB), or VX were used as OPNA. Established and experimental reactivators like the oximes obidoxime (OBI) and HI-6 or the non-oxime NOX-6 were added, and their effect was determined after 30 min. Depending on the reactivator properties, different experimental settings had to be chosen (continuous vs. discontinuous mode). The respective airway responses were investigated by video-microscopic imaging.

Determination of AChE activity in intact PCLS was performed for the first time. Exposure to the different OPNA led to low residual AChE activities (≤ 12±2 %) and small airway areas (≤ 13±4 %) comparable to previous in vivo data. GB-inhibited PCLS-AChE was reactivated best (OBI 83±6 % and HI-6 64±1 %), followed by VX (OBI 71±3 %, NOX-6 55±4 %, HI-6 51±1 %) and GF (OBI 13±3 % and HI-6 15±3 %). The data of airway area changes in PCLS after exposure to GB or GF and subsequential treatment with OBI were consistent with the previously reported values (OBI 74±6 % and HI-6 11±3 %). This suggests a good correlation between the AChE activities and the airway area data.

PCLS can be used for the determination of the AChE activity in intact tissue. Furthermore, they promote the concept of reduction of in vivo experiments. The results at the molecular (AChE activity) and functional level (airway area) were in line and matched previously published reactivation data of isolated rat red blood cell AChE. This shows that not only antimuscarinic drugs but also reactivators might play a role in the treatment of life-threatening airway constriction.

 

Figure 1: Method 1 – Using a photometer, the enzymatic Ellman assay, which is based on a colorimetric reaction, can be utilized to determine the AChE activity in intact PCLS. Method 2 – Video microscopic analysis of airway area changes in intact PCLS. Both methods are applied to observe the effects of different OPNAs and reactivators in intact PCLS – afterwards the results were correlated with each other.
Created with Biorender.com.

Second place

P01 • Sulfur Mustard-induced Senescence in Human Mesenchymal Stem Cells Influenced by Active Substance Candidates

Adrian Neu^a; Thilo Bertsche^b; Dirk Steinritz^a; Franz Worek^a; Annette Schmidt^c; Simone Rothmiller^a

The chemical warfare agent sulfur mustard (SM) is a bifunctional alkylating agent primarily harmful to the skin. SM exposure often results in chronic wound healing ­disorders. Human mesenchymal stem cells (hMSC) play an essential role in wound healing mechanisms, mainly due to their released cytokines. Therefore, a possible cause could be the SM-induced chronic senescence in hMSC since senescence inhibits their ability to regenerate in wound healing. The main negative effects of these senescent hMSC are persistence in damaged tissue for extended periods and maintaining an inflammatory milieu.

In this study, we investigated whether SM-induced senescence in hMSC can be ameliorated by the therapeutical drug candidates Poly (ADP-ribose) polymerase inhibitor Olaparib, the vitamin niacin or the fibroblast growth factor 2 (FGF-2) were investigated.

Isolation of hMSC was performed from the bone marrow of femoral heads. For quality assurance, differentiation potential, morphologies after differentiation, as well as surface antigens were verified by flow cytometry. Drug candidate treatment was performed 23 hours before (=^ prophylaxis) and 1 hour after (=^ therapy) SM exposure (40 µM). A senescence-associated β-galactosidase staining was performed to detect the percentage of senescent cells. Cytokine and chemokine secretion were examined in cell culture supernatants. NAD⁺ and ATP levels were quantified in Niacin treated hMSC and mRNA expression profiling was performed via qRT-PCR after FGF-2 treatment.

Olaparib induced senescence, whether used as therapy and prophylaxis, and was therefore not further investigated. Niacin and FGF-2 were capable of reducing SM induced senescence when administered prophylactically. An increased expression of TIMP1 by FGF-2 pre- and post-treatment as well as an increased downregulation of FGF-5 by FGF-2 pre-treatment was observed. SM-treated cells showed a reduced secretion of IL-6 after prophylactic administration of niacin. Additionally, increased intracellular NAD⁺ and ATP levels were shown after niacin pre-treatment.

In conclusion, niacin and FGF-2 pre-treatment ameliorated SM-induced senescence in hMSC. Further investigation should clarify the molecular mechanisms of action for both drug candidates in the context of SM-induced senescence in hMSC. Thus, FGF-2 and niacin may potentially serve as prophylactic agents for protection against SM exposure.

Figure 2: The microscopic images show SM and the control group with different treatments on day 21. Blue staining shows senescent cells according to a senescence-associated β-galactosidase assay. A reduction of senescence was observed in cells pretreated with niacin or FGF-2.

Third place

P32 • Reversal of Nerve Agent Toxicity Enabled by a Click-chemistry-derived Oxime Library – Reactivators of Butyrylcholinesterase

Tena Čadež; Zrinka Kovarik

Butyrylcholinesterase (BChE) is an enzyme capable of protecting synaptic acetylcholinesterase (AChE) from inhibition by bioscavenging organophosphorus nerve agents (OPNA; e.g. sarin, cyclosarin, tabun, VX) and is therefore a promising target for the development of therapies to counteract OP poisoning. In this study, we evaluated a click-chemistry-derived oxime library of over one hundred compounds as potential reactivators of BChE that had been phosphylated by OPNA. Interestingly, we identified reactivators for all four OP-BChE conjugates that were more efficient than the currently approved oximes in OP poisoning. Moreover, out of all tested oximes, a monopyridinium oximes showed high capacity in in vitro reactivation of inhibited BChE with various OPNA. In particular, the highest overall reactivation rate of almost 30,000 M-1 min-1 was determined for the reactivation of BChE phosphonylated with cyclosarin. As expected, our results indicated that the effectiveness of reactivation was highly dependent on the structure of both the OPNA-BChE conjugate and the oxime. However, it is quite unexpected that three 2-PAM analogues provided the most efficient recovery of BChE activity, although 2-PAM itself was a poor reactivator of BChE inhibited with OPNA. Out of the oxime library, triazole-annulated click-chemistry oximes were identified as potent reactivators of AChE inhibited by OPNA. The oxime-assisted BChE reactivation enabled the degradation of a 100-fold excess of cyclosarin in ex vivo conditions. Additionally, cytotoxicity profiles on neural cells revealed that post-treatment with the oxime-assisted catalytic BChE degradation of cyclosarin resulted in the preservation of 50 % of neural cells, while pre-treatment protected almost all of the cells, demonstrating that the tested oxime-assisted catalysis is viable for organophosphate (OP) bioscavenging in the blood.

Figure 3: New effective reactivators of inhibited BChE to provide the protection of synaptic AChE from OPNA inhibition by upgrading the BChE from a stoichiometric bioscavenger to an oxime-assisted bioscavenger.

Acknowledgments: The authors thank Professor K. Barry Sharpless and Professor Palmer Taylor for the long-term collaboration and the design, synthesis, and provision of the oxime libraries. This work was supported by the Croatian Science Foundation (IP-2018–01–7683).

Session 4: Innovative Approaches in Treatment of Organophosphate Poisoning (I)

P38 • Investigation on Interaction at the Orthosteric Epibatidine Binding Site of the Nicotinic Acetylcholine Receptor

Fabian Springer^a; Marian Freisleben^b; Sebastian ­Muschik^a; Antonia Brueser^a; Thomas Seeger^a; Dirk ­Steinritz^a; Franz Worek^a; Lorenz Meinel^c; Karin Veronika Niessen^a

Organophosphorus compounds (OP) inhibit the enzyme acetylcholinesterase (AChE). The following accumulation of acetylcholine and binding to the nicotinic acetylcholine receptors (nAChRs) leads to desensitization of the receptors. This dysfunction of the nAChRs results in a cholinergic crisis leading to death by respiratory arrest. Current therapeutics, comprising atropine and oximes are limited in their effectivity. Another approach is the use compounds that interact directly with the nAChR to restore its function, e.g. positive allosteric modulators. For effective screening of suitable agents, in vitro experiments such as affinity studies and bilayer-based electrophysiology with high-purity nAChR in biomimetic membranes are urgently needed.

nAChRs are ligand-gated, highly complex ion channels consisting of five subunits. Reasoning the need for huge amounts of highly standardized receptors, crude homogenized membrane fragments from Tetronarce californica (formerly Torpedo californica) electroplaques were used. Based on phylogenetic evolution, the muscle type nAChR shows a high degree of homology between species.

Figure 4: Nicotinic acetylcholine receptor with affinity and functionality data. A) Schematic representation of 2αβδγ muscle-type nAChR (Tetronarce californica, PDB 7QKO modified). The binding sites of channel blockers, positive allosteric modulators (PAMs) and the orthosteric binding site are highlighted. B) Affinity measurements were carried out on a modular pipetting platform. [³H]epibatidine was used as a marker for the orthosteric binding site. C) Highly purified nAChR fragments were embedded in biomimetic membranes after affinity purification. Addition of agonist carbamoylcholine leads to spontaneous opening events. Created with Biorender.com.

Crude cell membrane fragments containing nAChR were obtained using high-pressure homogenization. The unscalable process enables large, uniform batches. These could be used in parallel for further purification, as well as for different newly developed affinity and functionality assays. With these crude membrane fragments, saturation and competition assays have been developed for bindings studies at the orthosteric epibatidine binding sites. The scintillation proximity assay was performed on a custom-designed automated pipetting platform, with no radioactivity or toxic compounds handled openly.

For functionality assays using bilayer-based electrophysiology, high-purity nAChR fragments are needed due to the insertion of the receptor into biomimetic membranes. To obtain high-purity nAChR an affinity chromatographic method was developed. After upstream processing of the native tissue, the receptors were immobilized on nAChR antagonist α-bungarotoxin (α-Bgtx). Fractions were characterized using gel electrophoresis and immunoblotting.

In summary, the developed preparations of nAChR can be used for various new affinity and functionality assays to screen potential drug candidates that would be suitable for the treatment of nerve agent poisoning. Especially the combination of these methods is an effective tool to get insight into interactions on a molecular basis and to generate essential data for structure-activity relationships.

P57 • Molecular Chaperoning of nAChR through its Secretory Pathway: a Short Overview of Protein Networking

Sabrina Brockmöller; Karin Veronika Niessen; Thomas Seeger; Franz Worek

The current tense international situation emphasizes the ongoing threat posed by organophosphorus nerve agents (OPN). A mass-casualty incident would inevitably result in an overwhelmed medical treatment capacity. OPN act via irreversible inhibition of acetylcholinesterase followed by acetylcholine overflow at cholinergic synapses and potentially lethal cholinergic crisis. Hereby, the nicotinic acetylcholine receptor (nAChR) changes into a dysfunctional state. For the development of drugs able to restore nAChR function it is important to generate nAChR model systems, in particular, an expression system for the muscle type nAChR. The nAChR is a complex transmembrane protein with inefficient biogenesis. Its interaction with molecular chaperones during the secretory pathway ends in a wide range of protein networking. Understanding these interactions could allow the development of the desired nAChR model system.

This review looks at three special parts of the nAChR chaperoning, 1. endoplasmic reticulum (ER) which supports receptor subunits for folding, oligomerization and decreases degradation. nAChR associates with Calnexin, BiP direct and ERp57 indirect. Furthermore, BiP associates with other chaperones to control ER homeostasis. 2. Traffic and sorting chaperoning after biogenesis and leaving ER in COPII coated vesicles. In the secretory way between ERGIC and Golgi occurs a lot of control sorting of nAChR by chaperones for quality control. Some chaperones are capable to retain or to degrade nAChR. Uptake of nAChR into the Golgi apparatus and maturation is mediated by chaperones. 3. Chaperones mediate internalization and presentation of nAChR in plasma membranes. The suggested cascade of Agrin binding at muscle specific kinase (MuSK) and activation of signal transduction is only the first step in this mechanism.

From the overview of cellular targeting, trafficking and sorting it becomes evident that nAChR should not be considered alone when developing a cell model. Influencing some chaperones which associate directly or indirectly with nAChR might have a positive effect like increased fold yield. Supporting chaperones could result in a welfare cellular model system, which can handle the biogenesis and trafficking of nAChR.

Session 5: Innovative Approaches in Treatment of Organophosphate Poisoning (II)

T14 • Human HepaRG Liver Spheroids: Production, Cold Storage and Studies on Pyridinium Oxime-­induced Hepatotoxicity

Gabriele Horn; Franz Worek; Timo Wille

Case reports of organophosphorus compound (OP) ­poisoning addressed pathologic liver parameters in connection with oximes acting as reactivators of inhibited acetylcholinesterase. In this study, oxime-induced hepatotoxicity was investigated with a three-dimensional liver model. Storage of liver spheroids was optimized with tissue preservation solutions (TPS).

Liver spheroids were formed by combining HepaRG cells and human hepatic stellate cells. Cold storage of liver spheroids at 4 °C was investigated with a potassium chloride-rich TPS with/without additional iron chelator concentration and standard culture medium. Viability was assessed after rewarming with an ATP assay.

Drug-induced hepatotoxicity was assessed with diclofenac as positive hepatotoxic control, the oximes pralidoxime, obidoxime, HI-6, MMB-4 and the OP malathion or malaoxon alone or in the presence of obidoxime. Effective concentrations (EC₅₀) were determined with an ATP assay. The release of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and albumin secretion from liver spheroids were determined in supernatants.

Both TPS proved to be an appropriate tool to maintain viability for 72 hs without significant difference, whereas cold storage in standard culture medium resulted in a complete loss of viability.

All individually tested oximes and OP showed low cytotoxicity with EC₅₀ mainly > 2,000 μM. The combination of malaoxon with 1,000 µM obidoxime resulted in a significantly increased release of AST (77.7 ± 4 ng/mL for 2,000 µM malaoxon with 1,000 µM obidoxime vs. 31.8 ± 1 ng/mL with control) and a marked decrease of viability (EC₅₀ = 532 ± 33 µM) after 72 hs. Data is given as mean ± SEM.

Cold storage of liver spheroids can be optimized with specifically designed and commercially available TPS. Generally, the cytotoxicity of the individual oximes in liver spheroids is low and negligible at clinically relevant concentrations. The decrease of viability and the increased AST release after exposure to a combination of malaoxon with 1,000 μM obidoxime suggests that liver injury after OP poisoning may be triggered only if supratherapeutic oxime concentrations are used. These results will not impact the current therapy recommendations for OP poisoning.

Figure 5: Immunohistochemical study of liver spheroids: Overlay image of cytokeratin 8/18 (red), vimentin (green), and 4’,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 50 µm. More information in article WMM 2023; 67(3): 87–92.

T15 • Bioanalytical Procedures Proving Local Exposure of Human Hair to Nerve Agents and Vesicants

Harald John^a; Wolfgang Schmeißer^a; Tamara Lindl^b; ­Michael Schrader^b; Horst Thiermann^a

Chemical warfare agents are highly reactive compounds modifying proteins in vitro and in vivo. Sulfur mustard (SM) and similar vesicants alkylate, e.g., nucleophilic sulfur atoms of cysteine and methionine as well as oxygen atoms of the carboxylate group in glutamic acid residues. Organophosphorus nerve agents (OPNA) are known to phosphylate nucleophilic atoms in amino acid side chains, e.g., the oxygen atom in serine and tyrosine as well as the nitrogen atom in lysine residues [1]. Accordingly, adducted variants of plasma proteins like butyrylcholinesterase and serum albumin are formed after poison incorporation and its distribution. Thus, bioanalytical methods target such adducts to verify systemic exposure to certain agents [1].

Whereas several proteins from circulating blood are known as targets of adduction and may serve as precursors of peptide-adduct biomarkers [2][3], protein adducts documenting local exposure have not been described before. Therefore, we addressed human hair as a novel matrix for protein adduct analysis. A sample preparation procedure was elaborated including initial lysis of hair, subsequent precipitation of dissolved proteins and final proteolysis with ultrafiltration. Afterwards resulting cleavage products (alkylated peptides derived from SM exposure and pepsin-mediated proteolysis as well as phosphonylated single amino acids derived from exposure to the OPNA VX and sarin obtained after pronase treatment) were separated by micro liquid chromatography and detected by electrospray ionization tandem-mass spectrometry (µLC-ESI MS/MS) [4][5].

Based on sodium-dodecyl sulfate polyacryl gel electrophoresis (SDS-PAGE) combined with high-resolution tandem-mass spectrometry (MS/HR MS) of dissolved hair proteins, adducted type I and type II hard keratins were identified. These adducts of SM and OPNA were shown to be stable in dry hair for several weeks and months despite the influence of sunlight and air [4][5].

The presented methods might help to provide evidence for breaking of international law and the chemical weapons convention.

References:

[1] John et al. 19 (2021) J. Mass Spectrom. Adv. Clin. Lab 20–31; [2] John et al. Forensic Toxicol. 36 (2018) 61–71; [3] John et al. Arch. Toxicol. 93 (2019) 1881–1891; [4] Schmeißer et al. 96 (2022) Arch. Toxicol. 2287–2298; [5] John et al. Drug Test. Anal. (2023)

Figure 6: Chemical warfare agents modify hair protein structures detected for biomedical verification.

P50 • Disulfide-adducts in Human Serum Albumin Resulting from Exposure to Malodorants and their Identification

Paula Helena Sieber; Franz Worek; Harald John

Malodorants are used as non-lethal agents in military and civilian defense scenarios, and examples include sprays used as personal protection devices or riot control agents (RCA) [1–3]. These mixtures contain different organic compounds like alkyl thiols, which have olfactory properties and strong, repulsive characteristics [1]. When humans are exposed to these substances, they might suffer from headache, nausea, unconsciousness, coma or even fatal respiratory depression [5]. However, the deployment of RCA in warfare is banned by the Chemical Weapons Convention [5][6] and thus, bioanalytical procedures are needed for verification analysis. Accordingly, we present a robust mass spectrometry- (MS-) based method for the detection and identification of covalent malodorant-derived protein adducts. These adducts were formed during in vitro incubation of human plasma with ethyl mercaptan (SEt) forming disulfide-adducts with, e.g. Cys34 in human serum albumin (HSA). After proteolysis of adducted HSA, the dipeptide cysteine-proline (Cys
(-SEt)Pro) was detected using micro-liquid chromatography (µLC) coupled to high-resolution tandem-mass spectrometry (MS/HR MS). We also expect the formation of similar adducts with other malodorous thiols, which are potential biomarkers of exposure and useful for verification analysis for forensic purposes.

References:

[1] Timm and Reeves Disaster Manag. Resp. 5 (2007) 49–55; [2] Lewer and Davison Disarmament Forum 1 (2005) 37–51 UNIDIR/DF/2004/5; [3] Schmeisser et al. Army Res. Lab. (2013); [4] DHEW (NIOSH) Publication No 78–213 (1987); [5] Ellison, CRC Press (2007); [6] www.opcw.org

Figure 7: Formation of covalent disulfide-adducts with the side chain of Cys³⁴ of human serum albumin (HSA, left). Identification of disulfide-adducts by MS/HR MS (right).

 

Session 6: Advances in the Handling of Opioid Poisoning

T19 • Fentanyl and Derivatives – Opioids from a Police and Forensic Perspective

Björn Ahrens

Besides the relevance in organized crime, opioids are of particular concern in the eyes of security authorities, including when it comes to the use of carfentanil/remifentanil as an incapacitating agent, e.g. during the liberation of the hostages from the Moscow Dubrowka Theater.

Fentanyl is a well-known substance in cases of drug abuse and has accompanied forensic-chemical analysis of law enforcement laboratories for a while. Since about 2010, a consumer scene intensively looking for „legal highs“ has developed in Europe. Legal highs are substances that are not legally controlled but have analog pharamceutical properties as the controlled ones. Starting with cannabimimetic substances, quickly amphetamine-type stimulants, synthetic hallucinogens, and also opioids were found in seized materials by the police. An overview of Europe’s illegal drug market with a focus on fentanyl derivatives will be provided, including some of their impacts and drivers.

In 2018 German police forces arrested the largest distributor of carfentanil in Western Europe. During the crime scene investigation, approximately 800 g of pure carfentanil were seized. When presenting this event, the safety aspects for law enforcement and forensic personnel will be discussed to raise awareness of hazardous substances at crime scenes.

T21 • Pre-clinical Validation of Opioid Neutralising Antibodies that were Developed Using a Versatile Small Molecule Antibody Elicitation Technology

Rainer Wessel; Joseph Verdi

Fentanyl derivatives pose threats as chemical warfare agents. A notable example is carfentanil, a synthetic opioid that has already been weaponized and deployed during the 2002 Nord-Ort siege. The molecule was dispersed in an aerosolized form, causing hundreds of fatalities, the vast majority of which were civilian. The event highlights not only the lethality of fentanyl-like opioids but also the limitations of naloxone – the current standard of care for opioid overdose reversal. Several reports suggest that paramedics administered naloxone to a few individuals during the siege, however, unfortunately, the dissemination of the molecule was not effective enough. Naloxone is effective when administered successfully. However, its distribution is challenging in active combat scenarios. Further, naloxone has a remarkably short half-life and is an opioid itself, which generally limits its utility to the emergency post-overdose administration strategy. A pre-emptive overdose prevention strategy would be substantially more effective.

We sought to generate an opioid overdose-preventing antibody therapy. Antibodies have notably long half-lives, are well-tolerated as human therapeutics, and have well-understood biological characteristics. A carfentanil-binding antibody could be administered prophylactically to any individual prior to entering a scenario wherein exposure to carfentanil or related opioids is possible. Upon exposure, the already-present antibody would prevent toxicity by trapping the opioid in the bloodstream and thereby preventing it from entering the brain. To generate such an antibody, we have developed a versatile small-molecule antibody development platform. The platform, VAST, harnesses the famously-powerful immunogenicity of the African trypanosome parasite. By manipulating the major immunogenic trypanosome surface protein, we have created derivatized trypanosome membranes that retain their immunogenicity and can be fused to any molecule of interest through specific chemistry. The VAST is thus broadly applicable to any warfighter-relevant toxins, including fentanyl and related opioids. Immunizing mice with fentanyl-VAST yielded a series of monoclonal antibodies with picomolar fentanyl and carfentanil-binding affinity. Importantly, the antibodies are specific to fentanyl-like opioids and thus do not bind to naloxone or e.g., other pain-relief agents such as tramadol. The antibodies can completely prevent fentanyl and carfentanil toxicity in animal models, paving the way for clinical development.

Session 7: Innovative Approaches in Treatment of Poisoning by Blister and Lung Agents

T22 • Senescence after Sulfur Mustard Exposure as a Novel Pathomechanism of Wound Healing Disorders

Simone Rothmiller^a; Gabriele Horn^a; Catherine ­Schäfers^a; Niklas Jäger^a; Adrian Neu^a; Alexander Bürkle^b; Franz Worek^a; Dirk Steinritz^a; Annette Schmidt^c

Wound healing is a highly coordinated and complex process. Already small disturbances within this process may result in decreased healing, and chronic wounds are especially challenging to treat. Exposure to the alkylating warfare agent sulfur mustard (SM) is known to result in long-lasting wounds. Currently, the treatments are still insufficient and mostly supportive. The underlying cellular pathomechanisms are not yet fully understood. ­Senescence of key player cells in wound healing might affect healing due to the cells’ longevity and proinflammatory microenvironment. Thus, the main objective was identifying senescence as a new pathomechanism after SM exposure in wound healing-associated cell types.

Human mesenchymal stem cells (MSC) and human dermal fibroblasts (HDF) were exposed to SM, and senescence was determined by senescence-associated β-galactosidase (SA-β-gal) staining. Various additional senescence-associated markers were identified by quantitative real-time PCR and western blot. Migration was analyzed by IncuCyte. Senescence-associated secretory phenotype (SASP) was determined by Bio-Plex assay. Senolytics were applied, and cell viability was measured by XTT assay.

Senescence induction by SM was observed in MSC and HDF in a time- and concentration-dependent manner. Various senescence markers, such as SA-β-gal, gene expression changes in cell cycle regulation as well as DNA damage and repair pathway and SASP, were shown in both cell types. Senescence also reduced the migration ability of MSC. The senolytic drug ABT-263 significantly affected the viability of senescent cells more than of non-senescent cells and may thus reduce senescent cell burden.

Senescence after single-dose SM exposure in wound healing associated MSC and HDF was identified as a novel pathomechanism. Both cell types showed a variety of senescence markers, suggesting a common cellular answer to this stressor. These common changes could serve as potential new drug targets in treating SM-induced chronic wounds in a broader context.

Figure 8: Sulfur mustard (SM) exposure induces senescence in human mesenchymal stem cells and fibroblasts. Thus, they cannot replicate anymore and secrete a variety of proinflammatory factors, which might be a novel pathomechanism of wound healing disorders after SM exposure. Following, three interventions might open new therapeutic strategies: 1) Prevention of senescence, 2) Mitigation of negative effects, e.g. by interfering with secreted factors, or 3) Elimination of senescent cells. Created with Biorender.com.

P59 • Inhibition of Enzymes by Sulfur Mustard for the Development of a Rapid Test device

Daniel Barre; Dirk Steinritz; Franz Worek; Simone ­Rothmiller

Sulfur mustard (SM) is a chemical warfare agent banned by the Chemical Weapons Convention but has been used in several incidents in the past. As an alkylating agent, it reacts with biomacromolecules, including proteins. After a characteristic latency period, dermal exposure results in erythema, blistering, and ulcerations. A specific therapy does not exist. Thus, early and fast detection of SM followed by immediate decontamination is crucial. The most reliable methods often need substantial time, require complex sample preparation or demand high amounts of SM for reliable detection. A field-suitable and fast ­detection method, especially for the detection of small amounts of SM on the skin, is currently not available.
A possible approach could be developing a colorimetric test system based on SM-induced enzyme inhibition.

To find suitable enzyme candidates, the activity in the presence of SM was tested for multiple enzymes with commercially available colorimetric assays. Beta-lactamase was selected because it has a cysteine in its active site that might be targeted by SM. For the peroxidase, there were previously described alkylations with iodoacetate of multiple methionine residues that led to an inhibition. An enzyme solution in a buffer was prepared and incubated for the experiments with different sulfur mustard concentrations. After a defined pre-incubation of 25 minutes, the solutions were transferred into a 96-well plate, and enzyme activity was determined according to the manufacturer’s instructions. Absorbance was determined with a photometric plate reader at given wavelengths for one hour with measurements every minute.

Our findings show that SM inhibits the enzymes in a dose-dependent manner. The controls (EtOH, NaCl, HCl, and thiodiglycol) showed no or little influence on enzyme activity. Differences in enzyme activity were already visible to the naked eye.

These results suggest that the enzymes might help detect sulfur mustard. More research into the effect of temperature and other environmental factors on the enzymatic reaction is required to solidify its potential use as a detecting method.

P61 • Ferroptosis after Sulfur Mustard Exposure

Tobias Demel; Dirk Steinritz; Franz Worek; Simone ­Rothmiller

Even though the blistering agent sulfur mustard (SM) has been known for over 200 years, the exact mechanism of cellular damage is unknown. Thus, a specific therapy is missing. Since SM induces the production of reactive oxygen species and overloads anti-oxidative systems, oxidative stress is discussed as the main driver of toxicity. Initiated by failed antioxidant defense, the non-apoptotic and iron-dependent regulated cell death ferroptosis could be a new, until now not identified pathway in the molecular toxicology of SM. Targeting ferroptosis could represent a novel therapeutic option after SM exposition.

HaCaT or A549 cells were exposed to SM or ferroptosis inducers Erastin and RSL-3. Intensity changes of lipid droplets, glutathione peroxidase-4 (GPX4), system Xc- (xCT) and apoptosis inducing factor mitochondria associated 2 (AIFM2) were determined using flow cytometry. Effects of pretreatment and treatment with deferoxamine (DFO), ciclopirox (CPX), α-tocopherol (α-TOC), and ferrostatin-1 (FER-1) on cell viability were measured with Alamar Blue assay. Oxidized and reduced glutathione were measured with a luminescence-based assay.

GPX4 signal decreased after Erastin and RSL-3 but not SM exposure. Levels of xCT and AIFM2 were increased in both RSL-3 and SM-exposed cells. Lipid droplet staining showed higher intensity after RSL-3 and SM exposure but was lowered after Erastin. Treatment of SM-exposed cells with DFO and α-TOC reduced this effect. Total glutathione was reduced for RSL-3 and SM in a dose-depended manner, while both Erastin concentrations tested did not influence RSL-3. Cell viability at 300 μM SM exposure improved with 6.26 μM DFO and 10.23 μM α-TOC treatment. However, this effect was not significant for treatment and pretreatment in SM dose-response curves.

In conclusion, RSL-3, a GPX4-inhibitor, and SM seem to have overlapping effects on A549 and HaCaT cells. Erastin interfering with xCT might induce ferroptosis differently. Treatment with the literature-known ferroptosis inhibitors DFO, CPX, α-TOC, and FER-1 did not show significant effects on cell viability after SM exposure. More research in the context of SM and ferroptosis is needed to understand the underlying mechanisms.