Sulfur mustard alkylates steroid hormones and impacts hormone function in vitro

  • Robin LülingEmail author
  • Helena Singer
  • Tanja Popp
  • Harald John
  • Ingrid Boekhoff
  • Horst Thiermann
  • Lena J. Daumann
  • Konstantin Karaghiosoff
  • Thomas Gudermann
  • Dirk Steinritz
Toxicokinetics and Metabolism


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.


Betamethasone Dexamethasone Glucocorticoid Hydrocortisone Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) Nuclear magnetic resonance (NMR) Reporter gene assay 









2,5-Dihydroxybenzoic acid


Dulbecco’s modified Eagle’s medium




Firefly/Renilla dual-luciferase-assay




Fetal bovine serum






Glucocorticoid receptor


Glucocorticoid response element




Hydrochloric acid




Heat shock protein




Matrix-assisted laser desorption/ionization time-of-flight


Negative control


Nuclear magnetic resonance


Positive control




Room temperature


Real-time quantitative polymerase chain reaction


Sulfur mustard, bis-(2-chloroethyl) sulfide



Part of the work was supported by the German Research Foundation (Deutsche Forschungsgesellschaft, DFG, Research Training Group GRK 2338).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

204_2019_2571_MOESM1_ESM.docx (1.5 mb)
Supplementary material 1 (DOCX 1541 kb)


  1. Aktories K, Forth W, Förstermann U (2005) Allgemeine und spezielle Pharmakologie und Toxikologie: Für Studenten der Medizin, Veterinärmedizin, Pharmazie, Chemie und Biologie sowie für Ärzte, Tierärzte und Apotheker, 9., völlig überarb. Aufl. Elsevier Urban & Fischer, MünchenGoogle Scholar
  2. Asselin-Labat M-L, David M, Biola-Vidamment A, Lecoeuche D, Zennaro M-C, Bertoglio J, Pallardy M (2004) GILZ, a new target for the transcription factor FoxO3, protects T lymphocytes from interleukin-2 withdrawal-induced apoptosis. Blood 104(1):215–223. CrossRefGoogle Scholar
  3. Bielmann A, Sambiagio N, Wehr N, Gerber-Lemaire S, Bochet CG, Curty C (2018) Synthesis of different glutathione–sulfur mustard adducts of verified and potential biomarkers. RSC Adv 8(42):23881–23890. CrossRefGoogle Scholar
  4. Black RM (2008) An overview of biological markers of exposure to chemical warfare agents. J Anal Toxicol 32(1):2–9. CrossRefGoogle Scholar
  5. Black RM, Clarke RJ, Harrison JM, Read RW (1997) Biological fate of sulphur mustard: identification of valine and histidine adducts in haemoglobin from casualties of sulphur mustard poisoning. Xenobiotica 27(5):499–512. CrossRefGoogle Scholar
  6. Buschow SI, van Balkom BWM, Aalberts M, Heck AJR, Wauben M, Stoorvogel W (2010) MHC class II-associated proteins in B-cell exosomes and potential functional implications for exosome biogenesis. Immunol Cell Biol 88(8):851–856. CrossRefGoogle Scholar
  7. Deans AJ, West SC (2011) DNA interstrand crosslink repair and cancer. Nat Rev Cancer 11(7):467–480. CrossRefGoogle Scholar
  8. Dietmair S, Hodson MP, Quek L-E, Timmins NE, Gray P, Nielsen LK (2012) A multi-omics analysis of recombinant protein production in Hek293 cells. PLoS One 7(8):e43394. CrossRefGoogle Scholar
  9. Fidder A, Moes GWH, Scheffer AG, van der Schans GP, Baan RA, de Jong LPA, Benschop HP (1994) Synthesis, characterization, and quantitation of the major adducts formed between sulfur mustard and DNA of calf thymus and human blood. Chem Res Toxicol 7(2):199–204. CrossRefGoogle Scholar
  10. Fidder A, Noort D, de Jong AL, Trap HC, de Jong LP, Benschop HP (1996) Monitoring of in vitro and in vivo exposure to sulfur mustard by GC/MS determination of the N-terminal valine adduct in hemoglobin after a modified Edman degradation. Chem Res Toxicol 9(4):788–792. CrossRefGoogle Scholar
  11. Fu D, Calvo JA, Samson LD (2012) Balancing repair and tolerance of DNA damage caused by alkylating agents. Nat Rev Cancer 12(2):104–120. CrossRefGoogle Scholar
  12. Gandor F, Gawlik M, Thiermann H, John H (2015) Evidence of sulfur mustard exposure in human plasma by LC-ESI-MS-MS detection of the albumin-derived alkylated HETE-cp dipeptide and chromatographic investigation of its cis/trans isomerism. J Anal Toxicol 39(4):270–279. CrossRefGoogle Scholar
  13. Giri JG, Wells J, Dower SK, McCall CE, Guzman RN, Slack J, Bird TA, Shanebeck K, Grabstein KH, Sims JE (1994) Elevated levels of shed type II IL-1 receptor in sepsis. Potential role for type II receptor in regulation of IL-1 responses. J Immunol (Baltimore, Md. : 1950) 153(12):5802–5809Google Scholar
  14. Gråberg T, Strömmer L, Hedman E, Uzunel M, Ehrenborg E, Wikström A-C (2015) An ex vivo RT-qPCR-based assay for human peripheral leukocyte responsiveness to glucocorticoids in surgically induced inflammation. J Inflamm Res 8:149–160. CrossRefGoogle Scholar
  15. John H, Siegert M, Gandor F, Gawlik M, Kranawetvogl A, Karaghiosoff K, Thiermann H (2016) Optimized verification method for detection of an albumin-sulfur mustard adduct at Cys(34) using a hybrid quadrupole time-of-flight tandem mass spectrometer after direct plasma proteolysis. Toxicol Lett 244:103–111. CrossRefGoogle Scholar
  16. John H, Rychlik M, Thiermann H, Schmidt C (2018) Simultaneous quantification of atropine and scopolamine in infusions of herbal tea and Solanaceae plant material by matrix-assisted laser desorption/ionization time-of-flight (tandem) mass spectrometry. Rapid Commun Mass Spectrom RCM 32(22):1911–1921. CrossRefGoogle Scholar
  17. John H, Koller M, Worek F, Thiermann H, Siegert M (2019) Forensic evidence of sulfur mustard exposure in real cases of human poisoning by detection of diverse albumin-derived protein adducts. Arch Toxicol. Google Scholar
  18. Kehe K, Balszuweit F, Steinritz D, Thiermann H (2009) Molecular toxicology of sulfur mustard-induced cutaneous inflammation and blistering. Toxicology 263(1):12–19. CrossRefGoogle Scholar
  19. Kondo N, Takahashi A, Ono K, Ohnishi T (2010) DNA damage induced by alkylating agents and repair pathways. J Nucleic Acids 2010:543531. CrossRefGoogle Scholar
  20. Lang D, Knop J, Wesche H, Raffetseder U, Kurrle R, Boraschi D, Martin MU (1998) The type II IL-1 receptor interacts with the IL-1 receptor accessory protein: a novel mechanism of regulation of IL-1 responsiveness. J Immunol (Baltimore, Md. : 1950) 161(12):6871–6877Google Scholar
  21. Lawley PD, Brookes P (1967) Interstrand cross-linking of DNA by difunctional alkylating agents. J Mol Biol 25(1):143–160. CrossRefGoogle Scholar
  22. Lu NZ, Cidlowski JA (2005) Translational regulatory mechanisms generate N-terminal glucocorticoid receptor isoforms with unique transcriptional target genes. Mol Cell 18(3):331–342. CrossRefGoogle Scholar
  23. Mol MA, van der Schans GP, Lohman PH (1993) Quantification of sulfur mustard-induced DNA interstrand cross-links and single-strand breaks in cultured human epidermal keratinocytes. Mutat Res 294(3):235–245CrossRefGoogle Scholar
  24. Nicolaides NC, Galata Z, Kino T, Chrousos GP, Charmandari E (2010) The human glucocorticoid receptor: molecular basis of biologic function. Steroids 75(1):1–12. CrossRefGoogle Scholar
  25. Noort D, Hulst AG, de Jong LP, Benschop HP (1999) Alkylation of human serum albumin by sulfur mustard in vitro and in vivo: mass spectrometric analysis of a cysteine adduct as a sensitive biomarker of exposure. Chem Res Toxicol 12(8):715–721. CrossRefGoogle Scholar
  26. Noort D, Fidder A, Degenhardt-Langelaan CEAM, Hulst AG (2008) Retrospective detection of sulfur mustard exposure by mass spectrometric analysis of adducts to albumin and hemoglobin: an in vivo study. J Anal Toxicol 32(1):25–30CrossRefGoogle Scholar
  27. Okret S, Poellinger L, Dong Y, Gustafsson JA (1986) Down-regulation of glucocorticoid receptor mRNA by glucocorticoid hormones and recognition by the receptor of a specific binding sequence within a receptor cDNA clone. Proc Natl Acad Sci USA 83(16):5899–5903. CrossRefGoogle Scholar
  28. Pita R (2009) Toxin weapons: from World War I to jihadi terrorism. Toxin Rev 28(4):219–237. CrossRefGoogle Scholar
  29. Pita R, Anadón A (2015) Chemical Weapons of Mass Destruction and Terrorism. In: Gupta RC (ed) Handbook of toxicology of chemical warfare agents, 2nd edn. Elsevier/Academic Press, London, pp 55–65CrossRefGoogle Scholar
  30. Sacco O, Lantero S, Scarso L, Frangova V, Ottolini V, Rossi GA (1994) The increased expression of HLA-DR and ICAM-1 molecules by human bronchial epithelial cells, induced by activated mononuclear cells, is downregulated by nedocromil sodium. Mediators Inflamm 3(7):S7–S13. CrossRefGoogle Scholar
  31. Siegert M, Gandor F, Kranawetvogl A, Börner H, Thiermann H, John H (2019) Methionine329 in human serum albumin: a novel target for alkylation by sulfur mustard. Drug Test Anal 11(5):659–668. CrossRefGoogle Scholar
  32. Smith DE, Hanna R, Friend Della, Moore H, Chen H, Farese AM, MacVittie TJ, Virca GD, Sims JE (2003) The soluble form of IL-1 receptor accessory protein enhances the ability of soluble type II IL-1 receptor to inhibit IL-1 action. Immunity 18(1):87–96CrossRefGoogle Scholar
  33. Steinritz D, Striepling E, Rudolf K-D, Schröder-Kraft C, Püschel K, Hullard-Pulstinger A, Koller M, Thiermann H, Gandor F, Gawlik M, John H (2016) Medical documentation, bioanalytical evidence of an accidental human exposure to sulfur mustard and general therapy recommendations. Toxicol Lett 244:112–120. CrossRefGoogle Scholar
  34. Tsurufuji S, Sugio K, Takemasa F (1979) The role of glucocorticoid receptor and gene expression in the anti-inflammatory action of dexamethasone. Nature 280(5721):408–410. CrossRefGoogle Scholar
  35. Wu I, Shin SC, Cao Y, Bender IK, Jafari N, Feng G, Lin S, Cidlowski JA, Schleimer RP, Lu NZ (2013) Selective glucocorticoid receptor translational isoforms reveal glucocorticoid-induced apoptotic transcriptomes. Cell Death Dis 4:e453. CrossRefGoogle Scholar
  36. Xavier AM, Anunciato AKO, Rosenstock TR, Glezer I (2016) Gene expression control by glucocorticoid receptors during innate immune responses. Front Endocrinol 7:31. CrossRefGoogle Scholar
  37. Xu B, Zong C, Zhang Y, Zhang T, Wang X, Qi M, Wu J, Guo L, Wang P, Chen J, Liu Q, Xu H, Xie J, Zhang Z (2017) Accumulation of intact sulfur mustard in adipose tissue and toxicokinetics by chemical conversion and isotope-dilution liquid chromatography-tandem mass spectrometry. Arch Toxicol 91(2):735–747. CrossRefGoogle Scholar
  38. Yang K-J, Lin S-C, Huang S-J, Ching W-M, Hung C-H, Tzou D-LM (2014) Solid-state NMR study of fluorinated steroids. Steroids 80:64–70. CrossRefGoogle Scholar
  39. Young JC, Obermann WM, Hartl FU (1998) Specific binding of tetratricopeptide repeat proteins to the C-terminal 12-kDa domain of hsp90. J Biol Chem 273(29):18007–18010CrossRefGoogle Scholar
  40. Zubel T, Bürkle A, Mangerich A (2018) Mass spectrometric analysis of sulfur mustard-induced biomolecular adducts: are DNA adducts suitable biomarkers of exposure? Toxicol Lett 293:21–30. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Robin Lüling
    • 1
    • 2
    Email author
  • Helena Singer
    • 3
  • Tanja Popp
    • 2
    • 4
  • Harald John
    • 1
  • Ingrid Boekhoff
    • 2
  • Horst Thiermann
    • 1
  • Lena J. Daumann
    • 3
  • Konstantin Karaghiosoff
    • 3
  • Thomas Gudermann
    • 2
  • Dirk Steinritz
    • 1
    • 2
  1. 1.Bundeswehr Institute of Pharmacology and ToxicologyMunichGermany
  2. 2.Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität MünchenMunichGermany
  3. 3.Faculty for Chemistry and PharmacyLudwig-Maximilians-Universität MünchenMunichGermany
  4. 4.Bundeswehr Institute of RadiobiologyMunichGermany

Personalised recommendations