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Sulfur mustard alkylates steroid hormones and impacts hormone function in vitro

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.

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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

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Funding

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

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Correspondence to Robin Lüling.

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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

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Keywords

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