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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
  • 14 Downloads

Abstract

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.

Keywords

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

Abbreviations

BM

Betamethasone

CMV

Cytomegalovirus

DEX

Dexamethasone

DHB

2,5-Dihydroxybenzoic acid

DMEM

Dulbecco’s modified Eagle’s medium

DTT

Dithiothreitole

Dual-Glo

Firefly/Renilla dual-luciferase-assay

EtOH

Ethanol

FBS

Fetal bovine serum

Fluc

Firefly-luciferase

GC

Glucocorticoid

GCR

Glucocorticoid receptor

GRE

Glucocorticoid response element

HC

Hydrocortisone

HCl

Hydrochloric acid

HETE

Hydroxyl-ethyl-thio-ethyl

HSP

Heat shock protein

IL

Interleukine

MALDI-TOF

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

NC

Negative control

NMR

Nuclear magnetic resonance

PC

Positive control

P/S

Penicillin/streptomycin

RT

Room temperature

RT-qPCR

Real-time quantitative polymerase chain reaction

SM

Sulfur mustard, bis-(2-chloroethyl) sulfide

Notes

Funding

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)

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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

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