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Archives of Toxicology

, Volume 89, Issue 9, pp 1631–1643 | Cite as

Activation of the chemosensing transient receptor potential channel A1 (TRPA1) by alkylating agents

  • Bernhard Stenger
  • Franziska Zehfuß
  • Harald Mückter
  • Annette Schmidt
  • Frank Balszuweit
  • Eva Schäfer
  • Thomas Büch
  • Thomas Gudermann
  • Horst Thiermann
  • Dirk Steinritz
Organ Toxicity and Mechanisms

Abstract

The transient receptor potential ankyrin 1 (TRPA1) cation channel is expressed in different tissues including skin, lung and neuronal tissue. Recent reports identified TRPA1 as a sensor for noxious substances, implicating a functional role in the molecular toxicology. TRPA1 is activated by various potentially harmful electrophilic substances. The chemical warfare agent sulfur mustard (SM) is a highly reactive alkylating agent that binds to numerous biological targets. Although SM is known for almost 200 years, detailed knowledge about the pathophysiology resulting from exposure is lacking. A specific therapy is not available. In this study, we investigated whether the alkylating agent 2-chloroethyl-ethylsulfide (CEES, a model substance for SM-promoted effects) and SM are able to activate TRPA1 channels. CEES induced a marked increase in the intracellular calcium concentration ([Ca2+]i) in TRPA1-expressing but not in TRPA1-negative cells. The TRP-channel blocker AP18 diminished the CEES-induced calcium influx. HEK293 cells permanently expressing TRPA1 were more sensitive toward cytotoxic effects of CEES compared with wild-type cells. At low CEES concentrations, CEES-induced cytotoxicity was prevented by AP18. Proof-of-concept experiments using SM resulted in a pronounced increase in [Ca2+]i in HEK293-A1-E cells. Human A549 lung epithelial cells, which express TRPA1 endogenously, reacted with a transient calcium influx in response to CEES exposure. The CEES-dependent calcium response was diminished by AP18. In summary, our results demonstrate that alkylating agents are able to activate TRPA1. Inhibition of TRPA1 counteracted cellular toxicity and could thus represent a feasible approach to mitigate SM-induced cell damage.

Keywords

TRPA1 CEES Sulfur mustard Calcium signaling A549 

Abbreviations

AITC

Allyl isothiocyanate

AP18

4-(4-Chlorophenyl)-3-methylbut-3-en-2-oxime

AQ

Distilled water

[Ca2+]i

Intracellular calcium concentration

CEES

2-Chloroethyl-ethylsulfide

DMEM

Dulbecco’s modified eagle medium

DMSO

Dimethyl sulfoxide

ECL

Enhanced chemiluminescence

EtOH

Ethanol

FBS

Fetal bovine serum

h

Hours

HEK-A1-E; HEKA1

HEK293 cells, stable transfected with hTRPA1, clone E

HEK-WT; HEKWT

HEK293 wild-type cells

hTRPA1

Human transient receptor potential ankyrin 1

LC50

Lethal concentration, resulting in 50 % decreased cell viability in vitro

mA

Milliampere

mM

Millimolar

µM

Micromolar

min

Minutes

PBS

Phosphate-buffered saline

P/S

Penicillin–streptomycin

RIPA-buffer

Radio-immuno-precipitation-assay buffer

RR

Ruthenium red

s

Seconds

SD

Standard deviation

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEM

Standard error of the mean

SM

Sulfur mustard

TIH

Toxic inhalation hazard

TRPA1

Transient receptor potential ankyrin 1

V

Volt

WW

World War

Notes

Acknowledgments

We thank Vladimir Chubanov, Andreas Breit and Ram Prasad for their helpful support. This research was supported by the Transregional Collaborative Research Center 152, Project P15 and by a contract (E/UR2 W/CF504/CF560) of the German Armed Forces.

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

204_2014_1414_MOESM1_ESM.tif (127 kb)
Suppl. Figure 1 HEK293-A1-E cells were loaded with Fura-2 AM and stimulated with AITC or exposed to CEES. (A) As expected, 15 µM AITC stimulation (black squares) resulted in a distinct increase of 340/380 nm fluorescence ratio, indicating a pronounced calcium influx. Exposure of HEK293-A1-E cells to 10,000 µM (white triangles) or 3,333 µM CEES (gray circles) initially increased the 340/380 nm fluorescence ratio without concentration–response relationships or changes over time. (B) Zoom of (A): Moreover, the CEES-induced increase in Fura-2 AM fluorescence occurred even faster than in AITC-positive controls, suggesting chemical interference of CEES and Fura-2 AM. (C) 15 µM AITC stimulation had no influence on fluorescence emission at the isosbestic wavelength (360 nm), whereas even low concentrations of CEES (1,111-µm white triangles and 123-µM gray circles) showed a concentration-dependent decrease in fluorescence. This indicates a chemical interference of CEES with Fura-2 AM. HCl (1,000 µM, white diamonds) did not affect Fura-2 AM fluorescence at 360 nm, underlining our hypothesis of a CEES-induced Fura-2 AM modification. (TIFF 127 kb)
204_2014_1414_MOESM2_ESM.tif (79 kb)
Suppl. Figure 2 Acidification, i.e., decrease in pH values, following the hydrolysis of 10,000 µM CEES in MEM or distilled water (AQ). In AQ, almost immediate hydrolysis occurs, lowering the pH from 4.7 to 2.7. This corresponds to a 100x increase in proton concentration. After 30 min, the pH value decreased to 2.4 and remained almost unchanged afterward. In MEM, pH values decreased only slightly from 7.7 to 7.5 immediately after adding 10,000 µM CEES and to 7.0 after 30 or 60 min. The low concentration of free protons, present in MEM, was not even doubled, due to the buffer capacity of supplemented MEM. Horizontal bars represent significant changes (p < 0.05) between the groups. All experiments were conducted with n=3. Mean values ± S.D. are given. (TIFF 78 kb)
204_2014_1414_MOESM3_ESM.tif (140 kb)
Suppl. Figure 3 (A) Human lung epithelial cells (A549) were exposed to 2,500 µM CEES (white circles) or ethanol (solvent control, gray triangles), and increase in [Ca2+]i was assessed by aequorin luminescence. A549 cells showed a distinct calcium influx after CEES exposure. Ethanol had only negligible effects. All experiments were conducted with n=3. Mean values ± S.E.M. are given. (B) Pre-incubation of A549 with AP18 at various concentrations followed by a 2,500 µM CEES exposure resulted in a significant decrease in CEES-induced calcium influx. All experiments were conducted with n=3. Mean values ± S.E.M. are given. (C) Concentration–response relationship displaying peak luminescence values (shown in Suppl. Fig. 3B) revealed a concentration-dependent effect of AP18 on CEES-induced calcium influx in A549 cells. Although a distinct decrease in calcium influx was observed, a complete inhibition could not be achieved. All experiments were conducted with n=3. Mean values ± S.E.M. are given. (TIFF 140 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Bernhard Stenger
    • 1
  • Franziska Zehfuß
    • 1
  • Harald Mückter
    • 1
  • Annette Schmidt
    • 2
    • 3
  • Frank Balszuweit
    • 2
  • Eva Schäfer
    • 4
  • Thomas Büch
    • 4
  • Thomas Gudermann
    • 1
    • 5
    • 6
  • Horst Thiermann
    • 2
  • Dirk Steinritz
    • 1
    • 2
  1. 1.Walther-Straub-Institute of Pharmacology and ToxicologyLudwig-Maximilian-University MunichMunichGermany
  2. 2.Bundeswehr Institute of Pharmacology and ToxicologyMunichGermany
  3. 3.Department for Molecular and Cellular Sports MedicineGerman Sports University CologneCologneGermany
  4. 4.Independent Division of Clinical Pharmacology at Rudolf-Boehm-Institute for Pharmacology and ToxicologyUniversity of LeipzigLeipzigGermany
  5. 5.Comprehensive Pneumology Center Munich (CPC-M)German Center for Lung ResearchMunichGermany
  6. 6.DZHK (German Centre for Cardiovascular Research)Munich Heart AllianceMunichGermany

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