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


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.


TRPA1 CEES Sulfur mustard Calcium signaling A549 



Allyl isothiocyanate




Distilled water


Intracellular calcium concentration




Dulbecco’s modified eagle medium


Dimethyl sulfoxide


Enhanced chemiluminescence




Fetal bovine serum




HEK293 cells, stable transfected with hTRPA1, clone E


HEK293 wild-type cells


Human transient receptor potential ankyrin 1


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










Phosphate-buffered saline




Radio-immuno-precipitation-assay buffer


Ruthenium red




Standard deviation


Sodium dodecyl sulfate polyacrylamide gel electrophoresis


Standard error of the mean


Sulfur mustard


Toxic inhalation hazard


Transient receptor potential ankyrin 1




World War



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)


  1. Almers W, Neher E (1985) The Ca signal from fura-2 loaded mast cells depends strongly on the method of dye-loading. FEBS Lett 192(1):13–18. doi: 10.1016/0014-5793(85)80033-8 CrossRefPubMedGoogle Scholar
  2. Bandell M, Story GM, Hwang SW, Viswanath V, Eid SR, Petrus MJ, Earley TJ, Patapoutian A (2004) Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 41(6):849–857. doi: 10.1016/S0896-6273(04)00150-3 CrossRefPubMedGoogle Scholar
  3. Banner KH, Igney F, Poll C (2011) TRP channels: emerging targets for respiratory disease. Pharmacol Ther 130(3):371–384. doi: 10.1016/j.pharmthera.2011.03.005 CrossRefPubMedGoogle Scholar
  4. Bessac BF, Jordt S (2010) Sensory detection and responses to toxic gases: mechanisms, health effects, and countermeasures. Proc Am Thorac Soc 7(4):269–277. doi: 10.1513/pats.201001-004SM PubMedCentralCrossRefPubMedGoogle Scholar
  5. Bessac BF, Sivula M, von Hehn CA, Escalera J, Cohn L, Jordt S (2008) TRPA1 is a major oxidant sensor in murine airway sensory neurons. J Clin Investig 118(5):1899–1910. doi: 10.1172/JCI34192 PubMedCentralCrossRefPubMedGoogle Scholar
  6. Büch TR, Schäfer EA, Demmel M, Boekhoff I, Thiermann H, Gudermann T, Steinritz D, Schmidt A (2013) Functional expression of the transient receptor potential channel TRPA1, a sensor for toxic lung inhalants, in pulmonary epithelial cells. Chem Biol Interact 206(3):462–471. doi: 10.1016/j.cbi.2013.08.012 CrossRefPubMedGoogle Scholar
  7. Centers for Disease Control and Prevention (2003) Facts about sulfur mustard (
  8. de la Roche J, Eberhardt MJ, Klinger AB, Stanslowksy N, Wegner F, Koppert W, Reeh PW, Lampert A, Fischer MJM, Leffler A (2013) The molecular basis for species-specific activation of human TRPA1 by protons involves poorly conserved residues within transmembrane domains 5 and 6. J Biol Chem. doi: 10.1074/jbc.M113.479337 Google Scholar
  9. Defalco J, Steiger D, Gustafson A, Emerling DE, Kelly MG, Duncton MAJ (2010) Oxime derivatives related to AP18: agonists and antagonists of the TRPA1 receptor. Bioorg Med Chem Lett 20(1):276–279. doi: 10.1016/j.bmcl.2009.10.113 CrossRefPubMedGoogle Scholar
  10. Dons D (2013) As Syria crisis mounts, scientist looks back at last major chemical attack. Science 341(6150):1051. doi: 10.1126/science.341.6150.1051 CrossRefPubMedGoogle Scholar
  11. Fernandes ES, Fernandes MA, Keeble JE (2012) The functions of TRPA1 and TRPV1: moving away from sensory nerves. Br J Pharmacol 166(2):510–521. doi: 10.1111/j.1476-5381.2012.01851.x PubMedCentralCrossRefPubMedGoogle Scholar
  12. Finkelman FD (2014) Diesel exhaust particle exposure during pregnancy promotes development of asthma and atopy. J Allergy Clin Immunol. doi: 10.1016/j.jaci.2014.04.002 Google Scholar
  13. Gautam A, Vijayaraghavan R, Sharma M, Ganesan K (2006) Comparative toxicity studies of sulfur mustard (2,2′-dichloro diethyl sulfide) and monofunctional sulfur mustard (2-chloroethyl ethyl sulfide), administered through various routes in mice. J Med CBR Def 4.
  14. Gazdar AF, Oie HK, Shackleton CH, Chen TR, Triche TJ, Myers CE, Chrousos GP, Brennan MF, Stein CA, La Rocca RV (1990) Establishment and characterization of a human adrenocortical carcinoma cell line that expresses multiple pathways of steroid biosynthesis. Cancer Res 50:5488–5496PubMedGoogle Scholar
  15. Ghanei M, Rajaeinejad M, Motiei-Langroudi R, Alaeddini F, Aslani J (2011) Helium:oxygen versus air:oxygen noninvasive positive-pressure ventilation in patients exposed to sulfur mustard. Heart Lung 40(3):e84–e89. doi: 10.1016/j.hrtlng.2010.04.001 CrossRefPubMedGoogle Scholar
  16. Hinman A, Chuang H, Bautista DM, Julius D (2006) TRP channel activation by reversible covalent modification. Proc Natl Acad Sci USA 103(51):19564–19568. doi: 10.1073/pnas.0609598103 PubMedCentralCrossRefPubMedGoogle Scholar
  17. Hoenig SL (2002) Handbook of chemical warfare and terrorism. Greenwood Press, ConnecticutGoogle Scholar
  18. Jordt S, Bautista DM, Chuang H, McKemy DD, Zygmunt PM, Hogestatt ED, Meng ID, Julius D (2004) Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1. Nature 427(6971):260–265. doi: 10.1038/nature02282 CrossRefPubMedGoogle Scholar
  19. Kehe K, Balszuweit F, Steinritz D, Thiermann H (2009) Molecular toxicology of sulfur mustard-induced cutaneous inflammation and blistering. Toxicology 263(1):12–19. doi: 10.1016/j.tox.2009.01.019 CrossRefPubMedGoogle Scholar
  20. Kendall JM, Badminton MN (1998) Aequorea victoria bioluminescence moves into an exciting new era. Trends Biotechnol 16(5):216–224. doi: 10.1016/S0167-7799(98)01184-6 CrossRefPubMedGoogle Scholar
  21. Kinnamon SC (2012) Taste receptor signalling—from tongues to lungs. Acta Physiol (Oxf) 204(2):158–168. doi: 10.1111/j.1748-1716.2011.02308.x CrossRefGoogle Scholar
  22. Macpherson LJ, Dubin AE, Evans MJ, Marr F, Schultz PG, Cravatt BF, Patapoutian A (2007) Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines. Nature 445(7127):541–545. doi: 10.1038/nature05544 CrossRefPubMedGoogle Scholar
  23. McManus J, Huebner K (2005) Vesicants. Crit Care Clin 21(4):707–718 vi. doi:  10.1016/j.ccc.2005.06.005
  24. Mukhopadhyay I, Gomes P, Aranake S, Shetty M, Karnik P, Damle M, Kuruganti S, Thorat S, Khairatkar-Joshi N (2011) Expression of functional TRPA1 receptor on human lung fibroblast and epithelial cells. J Recept Signal Transduct Res 31(5):350–358. doi: 10.3109/10799893.2011.602413 CrossRefPubMedGoogle Scholar
  25. Nakatsuka K, Gupta R, Saito S, Banzawa N, Takahashi K, Tominaga M, Ohta T (2013) Identification of molecular determinants for a potent mammalian TRPA1 antagonist by utilizing species differences. J Mol Neurosci 51(3):754–762. doi: 10.1007/s12031-013-0060-2 CrossRefPubMedGoogle Scholar
  26. Nassini R, Pedretti P, Moretto N, Fusi C, Carnini C, Facchinetti F, Viscomi AR, Pisano AR, Stokesberry S, Brunmark C, Svitacheva N, McGarvey L, Patacchini R, Damholt AB, Geppetti P, Materazzi S, Guerrero-Hernandez A (2012) Transient receptor potential ankyrin 1 channel localized to non-neuronal airway cells promotes non-neurogenic inflammation. PLoS One 7(8):e42454. doi: 10.1371/journal.pone.0042454 PubMedCentralCrossRefPubMedGoogle Scholar
  27. Nilius B, Appendino G, Owsianik G (2012) The transient receptor potential channel TRPA1: from gene to pathophysiology. Pflug Arch 464(5):425–458. doi: 10.1007/s00424-012-1158-z CrossRefGoogle Scholar
  28. 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. doi: 10.1093/jat/32.1.25 CrossRefPubMedGoogle Scholar
  29. Pechura CM, Rall DP (1993) Veterans at risk: the health effects of mustard gas and lewisite. National Academy Press, Washington, DCGoogle Scholar
  30. Petrus M, Peier AM, Bandell M, Hwang SW, Huynh T, Olney N, Jegla T, Patapoutian A (2007) A role of TRPA1 in mechanical hyperalgesia is revealed by pharmacological inhibition. Mol Pain 3:40. doi: 10.1186/1744-8069-3-40 PubMedCentralCrossRefPubMedGoogle Scholar
  31. Pohanka M (2012) Antioxidants countermeasures against sulfur mustard. MRMC 12(8):742–748. doi: 10.2174/138955712801264783 CrossRefGoogle Scholar
  32. Ramsey IS, Delling M, Clapham DE (2006) An introduction to TRP channels. Annu Rev Physiol 68:619–647. doi: 10.1146/annurev.physiol.68.040204.100431 CrossRefPubMedGoogle Scholar
  33. Riccardi M (2003) Toxicological profile for sulfur mustard (UPDATE). U.S. Department of Health and Human Services 2003Google Scholar
  34. Rürup R, Schieder W, Kaufmann D (2000) Geschichte der Kaiser-Wilhelm-Gesellschaft im Nationalsozialismus. Wallstein, GöttingenGoogle Scholar
  35. Sawyer TW, Hamilton MG (2000) Effect of intracellular calcium modulation on sulfur mustard cytotoxicity in cultured human neonatal keratinocytes. Toxicol In Vitro 14(2):149–157CrossRefPubMedGoogle Scholar
  36. Schaefer EA, Stohr S, Meister M, Aigner A, Gudermann T, Buech TR (2013) Stimulation of the chemosensory TRPA1 cation channel by volatile toxic substances promotes cell survival of small cell lung cancer cells. Biochem Pharmacol 85(3):426–438. doi: 10.1016/j.bcp.2012.11.019 CrossRefPubMedGoogle Scholar
  37. Schmaltz F (2005) Kampfstoff-Forschung im Nationalsozialismus: Zur Kooperation von Kaiser-Wilhelm-Instituten, Militär und Industrie. Geschichte der Kaiser-Wilhelm-Gesellschaft im Nationalsozialismus, Bd. 11. Wallstein, GöttingenGoogle Scholar
  38. Shigetomi E, Jackson-Weaver O, Huckstepp RT, O’Dell TJ, Khakh BS (2013) TRPA1 channels are regulators of astrocyte basal calcium levels and long-term potentiation via constitutive d-serine release. J Neurosci 33(24):10143–10153. doi: 10.1523/JNEUROSCI.5779-12.2013 PubMedCentralCrossRefPubMedGoogle Scholar
  39. Shimomura O, Johnson FH, Morise H (1974) Mechanism of the luminescent intramolecular reaction of aequorin. Biochemistry 13(16):3278–3286. doi: 10.1021/bi00713a016 CrossRefPubMedGoogle Scholar
  40. Sidell FR, Urbanetti JS, Smith WJ, Hurst CG (eds) (1997) Medical aspects of chemical and biological warfare: chapter 7: vesicants. Office of the Surgeon General, Borden Institute, Walter Reed Army Medical Center Washington, DC. Office of The Surgeon General United States ArmyGoogle Scholar
  41. Simon SA, Liedtke W (2008) How irritating: the role of TRPA1 in sensing cigarette smoke and aerogenic oxidants in the airways. J Clin Investig. doi: 10.1172/JCI36111 Google Scholar
  42. Stewart CE (2006) Weapons of mass casualties and terrorism response handbook. Jones and Bartlett, Sudbury, MAGoogle Scholar
  43. Tewari-Singh N, Inturi S, Jain AK, Agarwal C, Orlicky DJ, White CW, Agarwal R, Day BJ (2014) Catalytic antioxidant AEOL 10150 treatment ameliorates sulfur mustard analog 2-chloroethyl ethyl sulfide-associated cutaneous toxic effects. Free Radic Biol Med 72:285–295. doi: 10.1016/j.freeradbiomed.2014.04.022 PubMedCentralCrossRefPubMedGoogle Scholar
  44. Thiermann H, Worek F, Kehe K (2013) Limitations and challenges in treatment of acute chemical warfare agent poisoning. Chem Biol Interact 206(3):435–443. doi: 10.1016/j.cbi.2013.09.015 CrossRefPubMedGoogle Scholar
  45. Veress LA, O’Neill HC, Hendry-Hofer TB, Loader JE, Rancourt RC, White CW (2010) Airway obstruction due to bronchial vascular injury after sulfur mustard analog inhalation. Am J Respir Crit Care Med 182(11):1352–1361. doi: 10.1164/rccm.200910-1618OC PubMedCentralCrossRefPubMedGoogle Scholar
  46. Wang YY, Chang RB, Allgood SD, Silver WL, Liman ER (2011) A TRPA1-dependent mechanism for the pungent sensation of weak acids. J Gen Physiol 137(6):493–505. doi: 10.1085/jgp.201110615 PubMedCentralCrossRefPubMedGoogle Scholar
  47. Wilson SR, Gerhold KA, Bifolck-Fisher A, Liu Q, Patel KN, Dong X, Bautista DM (2011) TRPA1 is required for histamine-independent, Mas-related G protein-coupled receptor-mediated itch. Nat Neurosci 14(5):595–602. doi: 10.1038/nn.2789 PubMedCentralCrossRefPubMedGoogle Scholar

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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