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.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Dulbecco’s modified Eagle’s medium
Fetal bovine serum
Glucocorticoid response element
Heat shock protein
Matrix-assisted laser desorption/ionization time-of-flight
Nuclear magnetic resonance
Real-time quantitative polymerase chain reaction
Sulfur mustard, bis-(2-chloroethyl) sulfide
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, München
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. https://doi.org/10.1182/blood-2003-12-4295
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. https://doi.org/10.1039/C8RA03360A
Black RM (2008) An overview of biological markers of exposure to chemical warfare agents. J Anal Toxicol 32(1):2–9. https://doi.org/10.1093/jat/32.1.2
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. https://doi.org/10.1080/004982597240460
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. https://doi.org/10.1038/icb.2010.64
Deans AJ, West SC (2011) DNA interstrand crosslink repair and cancer. Nat Rev Cancer 11(7):467–480. https://doi.org/10.1038/nrc3088
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. https://doi.org/10.1371/journal.pone.0043394
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. https://doi.org/10.1021/tx00038a013
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. https://doi.org/10.1021/tx9502150
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. https://doi.org/10.1038/nrc3185
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. https://doi.org/10.1093/jat/bkv010
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):5802–5809
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. https://doi.org/10.2147/JIR.S84165
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. https://doi.org/10.1016/j.toxlet.2015.09.027
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. https://doi.org/10.1002/rcm.8264
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. https://doi.org/10.1007/s00204-019-02461-2
Kehe K, Balszuweit F, Steinritz D, Thiermann H (2009) Molecular toxicology of sulfur mustard-induced cutaneous inflammation and blistering. Toxicology 263(1):12–19. https://doi.org/10.1016/j.tox.2009.01.019
Kondo N, Takahashi A, Ono K, Ohnishi T (2010) DNA damage induced by alkylating agents and repair pathways. J Nucleic Acids 2010:543531. https://doi.org/10.4061/2010/543531
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):6871–6877
Lawley PD, Brookes P (1967) Interstrand cross-linking of DNA by difunctional alkylating agents. J Mol Biol 25(1):143–160. https://doi.org/10.1016/0022-2836(67)90285-9
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. https://doi.org/10.1016/j.molcel.2005.03.025
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):235–245
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. https://doi.org/10.1016/j.steroids.2009.09.002
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. https://doi.org/10.1021/tx9900369
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):25–30
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. https://doi.org/10.1073/pnas.83.16.5899
Pita R (2009) Toxin weapons: from World War I to jihadi terrorism. Toxin Rev 28(4):219–237. https://doi.org/10.3109/15569540903246136
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 55–65
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. https://doi.org/10.1155/S0962935194000682
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. https://doi.org/10.1002/dta.2548
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):87–96
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. https://doi.org/10.1016/j.toxlet.2015.08.1105
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. https://doi.org/10.1038/280408a0
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. https://doi.org/10.1038/cddis.2012.193
Xavier AM, Anunciato AKO, Rosenstock TR, Glezer I (2016) Gene expression control by glucocorticoid receptors during innate immune responses. Front Endocrinol 7:31. https://doi.org/10.3389/fendo.2016.00031
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. https://doi.org/10.1007/s00204-016-1774-z
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. https://doi.org/10.1016/j.steroids.2013.11.020
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):18007–18010
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. https://doi.org/10.1016/j.toxlet.2017.12.014
Part of the work was supported by the German Research Foundation (Deutsche Forschungsgesellschaft, DFG, Research Training Group GRK 2338).
Conflict of interest
The authors declare no conflict of interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
About this article
Cite this article
Lüling, R., Singer, H., Popp, T. et al. Sulfur mustard alkylates steroid hormones and impacts hormone function in vitro. Arch Toxicol 93, 3141–3152 (2019). https://doi.org/10.1007/s00204-019-02571-x
- Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)
- Nuclear magnetic resonance (NMR)
- Reporter gene assay