Reduced Systemic Toxicity and Preserved Vestibular Toxicity Following Co-treatment with Nitriles and CYP2E1 Inhibitors: a Mouse Model for Hair Cell Loss

  • Sandra Saldaña-Ruíz
  • Pere Boadas-Vaello
  • Lara Sedó-Cabezón
  • Jordi LlorensEmail author
Research Article


Several nitriles, including allylnitrile and cis-crotononitrile, have been shown to be ototoxic and cause hair cell degeneration in the auditory and vestibular sensory epithelia of mice. However, these nitriles can also be lethal due in large part to the microsomal metabolic release of cyanide, which is mostly dependent on the activity of the 2E1 isoform of the cytochrome P450 (CYP2E1). In this study, we co-administered mice with a nitrile and, to reduce their lethal effects, a selective CYP2E1 inhibitor: diallylsulfide (DAS) or trans-1,2-dichloroethylene (TDCE). Both in female 129S1/SvImJ (129S1) mice co-treated with DAS and cis-crotononitrile and in male RjOrl:Swiss/CD-1 (Swiss) mice co-treated with TDCE and allylnitrile, the nitrile caused a dose-dependent loss of vestibular function, as assessed by a specific behavioral test battery, and of hair cells, as assessed by hair bundle counts using scanning electron microscopy. In the experiments, the CYP2E1 inhibitors provided significant protection against the lethal effects of the nitriles and did not diminish the vestibular toxicity as assessed by behavioral effects in comparison to animals receiving no inhibitor. Additional experiments using a single dose of allylnitrile demonstrated that TDCE does not cause hair cell loss on its own and does not modify the vestibular toxicity of the nitrile in either male or female 129S1 mice. In all the experiments, high vestibular dysfunction scores in the behavioral test battery predicted extensive to complete loss of hair cells in the utricles. This provides a means of selecting animals for subsequent studies of vestibular hair cell regeneration or replacement.


ototoxicity vestibular toxicity allylnitrile cis-crotononitrile mouse hair cell ablation 



We thank the student Xavier Farré for his assistance in some of the experiments, and Angel Messeguer (CSIC, Barcelona, Spain) for helping with the distillation of the cis-crotononitrile isomer. This work was supported by the Spanish Ministry of Science and Innovation (grant numbers BFU2009-06945 and BFU2012-31364) and the Generalitat of Catalonia (grant number 2009 SGR 1059). The SEM studies were performed at the Science and Technology Centers of the University of Barcelona.


  1. Balbuena E, Llorens J (2001) Behavioural disturbances and sensory pathology following allylnitrile exposure in rats. Brain Res 904:298–306PubMedCrossRefGoogle Scholar
  2. Balbuena E, Llorens J (2003) Comparison of cis- and trans-crotononitrile effects in the rat reveals specificity in the neurotoxic properties of nitrile isomers. Toxicol Appl Pharmacol 187:89–100PubMedCrossRefGoogle Scholar
  3. Barnes DW, Sanders VM, White KL Jr, Shopp GM Jr, Munson AE (1985) Toxicology of trans-1,2-dichloroethylene in the mouse. Drug Chem Toxicol 8:373–392PubMedCrossRefGoogle Scholar
  4. Boadas-Vaello P, Riera J, Llorens J (2005) Behavioral and pathological effects in the rat define two groups of neurotoxic nitriles. Toxicol Sci 88:456–466PubMedCrossRefGoogle Scholar
  5. Boadas-Vaello P, Jover E, Díez-Padrisa N, Bayona JM, Llorens J (2007) Differential role of CYP2E1-mediated metabolism in the lethal and vestibulotoxic effects of cis-crotononitrile in the mouse. Toxicol Appl Pharmacol 225:310–317PubMedCrossRefGoogle Scholar
  6. Boadas-Vaello P, Jover E, Saldaña-Ruíz S, Soler-Martín C, Chabbert C, Bayona JM, Llorens J (2009) Allylnitrile metabolism by CYP2E1 and other CYPs leads to distinct lethal and vestibulotoxic effects in the mouse. Toxicol Sci 107:461–472PubMedCrossRefGoogle Scholar
  7. Brady JF, Ishizaki H, Fukuto JM, Lin MC, Fadel A, Gapac JM, Yang CS (1991) Inhibition of cytochrome P-450 2E1 by diallyl sulfide and its metabolites. Chem Res Toxicol 4:642–647PubMedCrossRefGoogle Scholar
  8. Brugeaud A, Travo C, Demêmes D, Lenoir M, Llorens J, Puel J-L, Chabbert C (2007) Control of hair cell excitability by vestibular primary sensory neurons. J Neurosci 27:3503–3511PubMedCrossRefGoogle Scholar
  9. Brueckner JK, Ashby LP, Prichard JR, Porter JD (1999) Vestibulo-ocular pathways modulate extraocular muscle myosin expression patterns. Cell Tissue Res 295:477–484PubMedCrossRefGoogle Scholar
  10. Chanas B, Wang H, Ghanayem BI (2003) Differential metabolism of acrylonitrile to cyanide is responsible for the greater sensitivity of male vs female mice: role of CYP2E1 and epoxide hydrolases. Toxicol Appl Pharmacol 193:293–302PubMedCrossRefGoogle Scholar
  11. Chou SM, Hartmann HA (1964) Axonal lesions and waltzing syndrome after IDPN administration in rats. With a concept—“axostasis”. Acta Neuropathol 3:428–450PubMedCrossRefGoogle Scholar
  12. Crofton KM, Janssen R, Prazma J, Pulver S, Jr Barone S (1994) The ototoxicity of 3,3′-iminodipropionitrile: functional and morphological evidence of cochlear damage. Hear Res 80:129–140PubMedCrossRefGoogle Scholar
  13. Csillag A (2005) Atlas of the sensory organs. Humana, TotowaCrossRefGoogle Scholar
  14. Forge A, Schacht J (2000) Aminoglycoside antibiotics. Audiol Neurootol 5:3–22PubMedCrossRefGoogle Scholar
  15. Gagnaire F, Marignac B, Ban M, Langlais C (2001) The ototoxic effects induced in rats by treatment for 12 weeks with 2-butenenitrile, 3-butenenitrile and cis-2-pentenenitrile. Pharmacol Toxicol 88:126–134PubMedCrossRefGoogle Scholar
  16. Golub JS, Tong L, Ngyuen TB, Hume CR, Palmiter RD, Rubel EW, Stone JS (2012) Hair cell replacement in adult mouse utricles after targeted ablation of hair cells with diphteria toxin. J Neurosci 32:15093–15105Google Scholar
  17. Griffin JW, Hoffman PN, Clark AW, Carroll PT, Price DL (1978) Slow axonal transport of neurofilament proteins: impairment of beta, beta′-iminodipropionitrile administration. Science 202:633–635PubMedCrossRefGoogle Scholar
  18. Heydt JL, Cunningham LL, Rubel EW, Coltrera MD (2004) Round window gentamicin application: an inner ear hair cell damage protocol for the mouse. Hear Res 192:65–74PubMedCrossRefGoogle Scholar
  19. Ishibashi T, Takumida M, Akagi N, Hirakawa K, Anniko M (2009) Changes in transient receptor potential vanilloid (TRPV) 1, 2, 3 and 4 expression in mouse inner ear following gentamicin challenge. Acta Otolaryngol 129:116–126PubMedCrossRefGoogle Scholar
  20. Khan Z, Carey J, Park HJ, Lehar M, Lasker D, Jinnah HA (2004) Abnormal motor behavior and vestibular dysfunction in the stargazer mouse mutant. Neuroscience 127:785–796PubMedCrossRefGoogle Scholar
  21. Lilly PD, Thornton-Manning JR, Gargas ML, Clewell HJ, Andersen ME (1998) Kinetic characterization of CYP2E1 inhibition in vivo and in vitro by the chloroethylenes. Arch Toxicol 72:609–621PubMedCrossRefGoogle Scholar
  22. Llorens J, Demêmes D (1996) 3,3′-Iminodipropionitrile induces neurofilament accumulations in the perikarya of rat vestibular ganglion neurons. Brain Res 717:118–126PubMedCrossRefGoogle Scholar
  23. Llorens J, Demêmes D, Sans A (1993) The behavioral syndrome caused by 3,3′-iminodipropionitrile and related nitriles in the rat is associated with degeneration of the vestibular sensory hair cells. Toxicol Appl Pharmacol 123:199–210PubMedCrossRefGoogle Scholar
  24. Llorens J, Demêmes D (1994) Hair cell degeneration resulting from 3,3′-iminodipropionitrile toxicity in the rat vestibular epithelia. Hear Res 76:78–86PubMedCrossRefGoogle Scholar
  25. Llorens J, Rodríguez-Farré E (1997) Comparison of behavioral, vestibular, and axonal effects of subchronic IDPN in the rat. Neurotoxicol Teratol 19:117–127PubMedCrossRefGoogle Scholar
  26. Llorens J, Aguiló A, Rodríguez-Farré E (1998) Behavioral disturbances and vestibular pathology following crotonitrile exposure in rats. J Periph Nerv Sys 3:189–196Google Scholar
  27. Mathews JM, Etheridge AS, Raymer JH, Black SR, Pulliam DW Jr, Bucher JR (1998) Selective inhibition of cytochrome P450 2E1 in vivo and in vitro with trans-1,2-dichloroethylene. Chem Res Toxicol 11:778–785PubMedCrossRefGoogle Scholar
  28. Murillo-Cuesta S, Contreras J, Cediel R, Varela-Nieto I (2009) Comparison of different aminoglycoside antibiotic treatments to refine ototoxicity studies in adult mice. Lab Anim 44:124–131PubMedCrossRefGoogle Scholar
  29. Oesterle EC, Campbell S, Taylor RR, Forge A, Hume CR (2008) Sox2 and JAGGED1 expression in normal and drug-damaged adult mouse inner ear. J Assoc Res Otolaryngol 9:65–89PubMedCrossRefGoogle Scholar
  30. Saldaña-Ruíz S, Soler-Martín C, Llorens J (2012a) Role of CYP2E1-mediated metabolism in the acute and vestibular toxicities of nineteen nitriles in the mouse. Toxicol Lett 208:125–132PubMedCrossRefGoogle Scholar
  31. Saldaña-Ruíz S, Hernández-Mir G, Sedó-Cabezón L, Cutillas B, Llorens J (2012b) Vestibular toxicity of cis-2-pentenenitrile in the rat. Toxicol Lett 211:281–288PubMedCrossRefGoogle Scholar
  32. Schlecker C, Praetorius M, Brough DE, Presler RG Jr, Hsu C, Plinkert PK, Staecker H (2011) Selective atonal gene delivery improves balance function in a mouse model of vestibular disease. Gene Ther 18:884–890PubMedCrossRefGoogle Scholar
  33. Seoane A, Demêmes D, Llorens J (2001a) Relationship between insult intensity and mode of hair cell loss in the vestibular system of rats exposed to 3,3′-iminodipropionitrile. J Comp Neurol 439:385–399PubMedCrossRefGoogle Scholar
  34. Seoane A, Demêmes D, Llorens J (2001b) Pathology of the rat vestibular sensory epithelia during subchronic 3,3′-iminodipropionitrile exposure: hair cells may not be the primary target of toxicity. Acta Neuropathol 102:339–348PubMedGoogle Scholar
  35. Soler-Martín C, Diez-Padrisa N, Boadas-Vaello P, Llorens J (2007) Behavioral disturbances and hair cell loss in the inner ear following nitrile exposure in mice, guinea pigs, and frogs. Toxicol Sci 96:123–132PubMedCrossRefGoogle Scholar
  36. Soler-Martín C, Boadas-Vaello P, Saldaña-Ruíz S, Cutillas B, Llorens J (2011) Butenenitriles have low axonopathic potential in the rat. Toxicol Lett 200:187–193PubMedCrossRefGoogle Scholar
  37. Taylor RR, Nevill G, Forge A (2008) Rapid hair cell loss: a mouse model for cochlear lesions. J Assoc Res Otolaryngol 9:44–64PubMedCrossRefGoogle Scholar

Copyright information

© Association for Research in Otolaryngology 2013

Authors and Affiliations

  • Sandra Saldaña-Ruíz
    • 1
    • 2
  • Pere Boadas-Vaello
    • 3
  • Lara Sedó-Cabezón
    • 1
    • 2
  • Jordi Llorens
    • 1
    • 2
    Email author
  1. 1.Departament de Ciències Fisiològiques IIUniversitat de BarcelonaCataloniaSpain
  2. 2.Institut d’Investigació Biomèdica de Bellvitge (IDIBELL)CataloniaSpain
  3. 3.NEOMA Research Group, Departament de Ciències Mèdiques, Facultat de MedicinaUniversitat de GironaCataloniaSpain

Personalised recommendations