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

John, Harald; Koller, Marianne; Worek, Franz; Thiermann, Horst; Siegert, Markus

doi:10.1007/s00204-019-02461-2

Cited by 40 publications

(68 citation statements)

References 20 publications

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“…Despite the chemical weapons convention (CWC), which prohibits the development, production, storage, and deployment of CWAs, it has been deployed in various conflicts in the last decades 1–6 . Most recently, SM was used by the terrorist group islamic state of Iraq and the Levant (ISIL) in Syria and Northern Iraq, 3–6 and accidental exposures have also been reported, 7,8 thus underlining the need to understand the pathophysiology, to develop appropriate antidotes, and to establish analytical methods for biomedical verification of poisoning. Once SM enters the bloodstream, it forms adducts with various molecules by alkylation of, for example, proteins, DNA, hormones, glutathione, and N ‐acetylcysteine 9–14 .…”

Section: Introductionmentioning

confidence: 99%

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

Lüling

Schmeißer²,

Siegert

et al. 2020

Drug Testing and Analysis

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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 Cys282 of CK and Cys232 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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Section: Introductionmentioning

confidence: 99%

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

Lüling

Schmeißer²,

Siegert

et al. 2020

Drug Testing and Analysis

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“…While secondary markers are not unambiguous for identification but able to support identifications based on primary ones, data has to be reported from at least one primary biomarker. For the identification of SM in human plasma samples, the primary biomarkers are HETE-CP (from HSA), HETE-CPF (from HSA), N1/N3-HETE-His (from HSA and Globin), and other protein-or amino acid-adducts (e.g., AE 230 (-HETE) VSKL from HSA [12] and LGM 329 (-HETE) F from HSA, [9]), while the secondary marker for SM is thiodiglycol [41]. Our aim was to develop methods for the analysis and identification of HETETE-CP and HETETE-CPF as primary biomarkers of Q in plasma.…”

Section: Resultsmentioning

confidence: 99%

“…Q and SM competed for the thiol groups of a limited amount of Cys 34 applied as neat HSA instead of plasma to minimize any side reactions with other plasma proteins. As numerous amino acids of HSA are expected to be prone to alkylation as exemplarily shown for Glu 230 [12] and Met 329 [9], a low HSA concentration was applied. After incubation, samples were subjected to pronase-mediated proteolysis to monitor HETE-CP and HETETE-CP simultaneously as measures of their respective HSA-adducts.…”

Section: Alkylationmentioning

confidence: 99%

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

Blum¹,

Richter

Siegert

et al. 2020

Anal Bioanal Chem

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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 Cys34 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 Cys34. Furthermore, the results of molecular dynamics simulations are indicative that the protein environment around Cys34 allows adduct formation with elongated but not bulky molecules such as Q, and identify important hydrogen bonding interactions and hydrophobic contacts.

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“…The implementation of the Chemical Weapons Convention (CWC) in 1997 [ 1 ] was a milestone in the prohibition of chemical warfare agents (CWA). However, the repeated use of CWA in military conflicts such as in Syria, by terrorists and against individuals, underlines the ongoing threat to the population [ 2 , 3 , 4 , 5 ] and demands further research on the biological properties of CWA and improved medical countermeasures against these agents.…”

Section: Introductionmentioning

confidence: 99%

In Vitro Interaction of Organophosphono- and Organophosphorothioates with Human Acetylcholinesterase

et al. 2020

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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 P-alkyl, P-O-alkyl and N,N-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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Forensic evidence of sulfur mustard exposure in real cases of human poisoning by detection of diverse albumin-derived protein adducts

Cited by 40 publications

References 20 publications

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

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

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

In Vitro Interaction of Organophosphono- and Organophosphorothioates with Human Acetylcholinesterase

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