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.
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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
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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.
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204_2014_1414_MOESM1_ESM.tif
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
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
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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Stenger, B., Zehfuß, F., Mückter, H. et al. Activation of the chemosensing transient receptor potential channel A1 (TRPA1) by alkylating agents. Arch Toxicol 89, 1631–1643 (2015). https://doi.org/10.1007/s00204-014-1414-4
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DOI: https://doi.org/10.1007/s00204-014-1414-4