Archives of Toxicology

, Volume 93, Issue 11, pp 3291–3303 | Cite as

Novel insights into the mechanism of cyclophosphamide-induced bladder toxicity: chloroacetaldehyde’s contribution to urothelial dysfunction in vitro

  • Kylie A. Mills
  • Russ Chess-Williams
  • Catherine McDermottEmail author
Organ Toxicity and Mechanisms


The clinical use of cyclophosphamide and ifosfamide is limited by a resultant bladder toxicity which has been attributed to the metabolite acrolein. Another metabolite chloroacetaldehyde (CAA) associated with nephrotoxicity, has not been investigated for toxicity in the bladder and this study investigates the effects of acrolein and CAA on human urothelial cells in vitro. Human urothelial cells (RT4 and T24) were treated with acrolein or CAA and changes in cell viability, reactive oxygen species, caspase-3 activity and release of urothelial mediators ATP, acetylcholine, PGE2 were measured. The protective effects of N-acetyl cysteine (NAC) were also assessed. Both metabolites were toxic to human urothelial cells, however, CAA significantly decreased cell viability at a ten-fold lower concentration (10 µM) than acrolein (100 µM). This was associated with increased ROS production and caspase-3 activity. NAC protected cells from these changes. In RT4 cells 100 µM acrolein caused a significant increase in basal and stretch-induced ATP, Ach and PGE2 release. In T24 cells chloroacetaldehyde (10 µM) increased basal and stimulated ATP and PGE2 levels. Again, NAC protected against changes in urothelial mediator release following acrolein or CAA. This study is the first to report that CAA in addition to acrolein contributes to the urotoxicity of cyclophosphamide and ifosfamide. Both metabolites altered urothelial mediator levels which could contribute to the sensory and functional bladder changes experienced by patients after treatment with cyclophosphamide or ifosfamide. Alterations in urothelial cell viability and mediator release may be causally linked to oxidative stress, with NAC providing protection against these changes.


Cyclophosphamide Chloroacetaldehyde Acrolein Ifosfamide Urothelium Urotoxicity Oxidative stress N-acetyl cysteine 



This work was funded by the Cancer Council Queensland (Grant number 1032032).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Al-Rawithi S, El-Yazigi A, Ernst P, Al-Fiar F, Nicholls PJ (1998) Urinary excretion and pharmacokinetics of acrolein and its parent drug cyclophosphamide in bone marrow transplant patients. Bone Marrow Transplant 22(5):485–490. CrossRefPubMedGoogle Scholar
  2. Anoopkumar-Dukie S, Carey JB, Conere T, O’Sullivan E, van Pelt FN, Allshire A (2005) Resazurin assay of radiation response in cultured cells. Br J Radiol 78(934):945–947. CrossRefPubMedGoogle Scholar
  3. Aronsson P, Johnsson M, Vesela R, Winder M, Tobin G (2012) Adenosine receptor antagonism suppresses functional and histological inflammatory changes in the rat urinary bladder. Auton Neurosci 171(1–2):49–57. CrossRefPubMedGoogle Scholar
  4. Beauchamp RO Jr, Andjelkovich DA, Kligerman AD, Morgan KT, Heck HD (1985) A critical review of the literature on acrolein toxicity. Crit Rev Toxicol 14(4):309–380. CrossRefPubMedGoogle Scholar
  5. Benesic A, Schwerdt G, Hennemeier I, Sauvant C, Mildenberger S, Gekle M (2014) The nephrotoxic Ifosfamide-metabolite chloroacetaldehyde interferes with renal extracellular matrix homeostasis. Cell Physiol Biochem 33(4):1106–1116. CrossRefPubMedGoogle Scholar
  6. Birder LA (2010) Urothelial signaling. Auton Neurosci 153(1–2):33–40. CrossRefPubMedGoogle Scholar
  7. Birder L, Andersson KE (2013) Urothelial signaling. Physiol Rev 93(2):653–680. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Birder LA, Barrick SR, Roppolo JR et al (2003) Feline interstitial cystitis results in mechanical hypersensitivity and altered ATP release from bladder urothelium. Am J Physiol Renal Physiol 285(3):F423–F429. CrossRefPubMedGoogle Scholar
  9. Birder LA, Kanai AJ, Cruz F, Moore K, Fry CH (2010) Is the urothelium intelligent? Neurourol Urodyn 29(4):598–602. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Biswal S, Acquaah-Mensah G, Datta K, Wu X, Kehrer JP (2002) Inhibition of cell proliferation and AP-1 activity by acrolein in human A549 lung adenocarcinoma cells due to thiol imbalance and covalent modifications. Chem Res Toxicol 15(2):180–186CrossRefGoogle Scholar
  11. Brock N, Stekar J, Pohl J, Niemeyer U, Scheffler G (1979) Acrolein, the causative factor of urotoxic side-effects of cyclophosphamide, ifosfamide, trofosfamide and sufosfamide. Arzneimittelforschung 29(4):659–661PubMedGoogle Scholar
  12. Burnstock G (2009) Purinergic mechanosensory transduction and visceral pain. Mol Pain 5:69. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Burnstock G (2011) Therapeutic potential of purinergic signalling for diseases of the urinary tract. BJU Int 107(2):192–204. CrossRefPubMedGoogle Scholar
  14. Chen N, Aleksa K, Woodland C, Rieder M, Koren G (2007) The effect of N-acetylcysteine on ifosfamide-induced nephrotoxicity: in vitro studies in renal tubular cells. Transl Res 150(1):51–57. CrossRefPubMedGoogle Scholar
  15. Chen N, Aleksa K, Woodland C, Rieder M, Koren G (2008) N-Acetylcysteine prevents ifosfamide-induced nephrotoxicity in rats. Br J Pharmacol 153(7):1364–1372. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Coggins PR, Ravdin RG, Eisman SH (1960) Clinical evaluation of a new alkylating agent: cytoxan (cyclophosphamide). Cancer 13:1254–1260CrossRefGoogle Scholar
  17. Daly DM, Chess-Williams R, Chapple C, Grundy D (2010) The inhibitory role of acetylcholine and muscarinic receptors in bladder afferent activity. Eur Urol 58(1):22–28. (discussion 31–2) CrossRefPubMedGoogle Scholar
  18. Dang K, Lamb K, Cohen M, Bielefeldt K, Gebhart GF (2008) Cyclophosphamide-induced bladder inflammation sensitizes and enhances P2X receptor function in rat bladder sensory neurons. J Neurophysiol 99(1):49–59. CrossRefPubMedGoogle Scholar
  19. de Groat WC, Yoshimura N (2001) Pharmacology of the lower urinary tract. Annu Rev Pharmacol Toxicol 41:691–721. CrossRefPubMedGoogle Scholar
  20. de Jongh R, van Koeveringe GA, van Kerrebroeck PE, Markerink-van Ittersum M, de Vente J, Gillespie JI (2007) The effects of exogenous prostaglandins and the identification of constitutive cyclooxygenase I and II immunoreactivity in the normal guinea pig bladder. BJU Int 100(2):419–429. CrossRefPubMedGoogle Scholar
  21. de Jongh R, Grol S, van Koeveringe GA, van Kerrebroeck PE, de Vente J, Gillespie JI (2009) The localization of cyclo-oxygenase immuno-reactivity (COX I-IR) to the urothelium and to interstitial cells in the bladder wall. J Cell Mol Med 13(9B):3069–3081. CrossRefPubMedGoogle Scholar
  22. Deberry JJ, Schwartz ES, Davis BM (2014) TRPA1 mediates bladder hyperalgesia in a mouse model of cystitis. Pain. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Fukuoka M, Negoro S, Masuda N et al (1991) Placebo-controlled double-blind comparative study on the preventive efficacy of mesna against ifosfamide-induced urinary disorders. J Cancer Res Clin Oncol 117(5):473–478CrossRefGoogle Scholar
  24. Golubeva AV, Zhdanov AV, Mallel G, Dinan TG, Cryan JF (2014) The mouse cyclophosphamide model of bladder pain syndrome: tissue characterization, immune profiling, and relationship to metabotropic glutamate receptors. Physiol Rep 2(3):e00260. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Goren MP, Wright RK, Pratt CB, Pell FE (1986) Dechloroethylation of ifosfamide and neurotoxicity. Lancet 2(8517):1219–1220CrossRefGoogle Scholar
  26. Gurbuz N, Ozkul A, Burgaz S (2009) Effects of vitamin C and N-acetylcysteine against cyclophosphamide-induced genotoxicity in exfoliated bladder cells of mice in vivo. J BUON 14(4):647–652PubMedGoogle Scholar
  27. Hanly L, Figueredo R, Rieder MJ, Koropatnick J, Koren G (2012) The Effects of N-acetylcysteine on ifosfamide efficacy in a mouse xenograft model. Anticancer Res 32(9):3791–3798PubMedGoogle Scholar
  28. Hanna-Mitchell AT, Birder LA (2008) New insights into the pharmacology of the bladder. Curr Opin Urol 18(4):347–352. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Hawthorn MH, Chapple CR, Cock M, Chess-Williams R (2000) Urothelium-derived inhibitory factor(s) influences on detrusor muscle contractility in vitro. Br J Pharmacol 129(3):416–419. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Horton ND, Biswal SS, Corrigan LL, Bratta J, Kehrer JP (1999) Acrolein causes inhibitor kappaB-independent decreases in nuclear factor kappaB activation in human lung adenocarcinoma (A549) cells. J Biol Chem 274(14):9200–9206CrossRefGoogle Scholar
  31. Iijima K, De Wachter S, Wyndaele JJ (2007) Effects of the M3 receptor selective muscarinic antagonist darifenacin on bladder afferent activity of the rat pelvic nerve. Eur Urol 52(3):842–847. CrossRefPubMedGoogle Scholar
  32. Jo GH, Kim GY, Kim WJ, Park KY, Choi YH (2014) Sulforaphane induces apoptosis in T24 human urinary bladder cancer cells through a reactive oxygen species-mediated mitochondrial pathway: the involvement of endoplasmic reticulum stress and the Nrf2 signaling pathway. Int J Oncol 45(4):1497–1506. CrossRefPubMedGoogle Scholar
  33. Joqueviel C, Malet-Martino M, Martino R (1997) A 13C NMR study of 2-(13)C-chloroacetaldehyde, a metabolite of ifosfamide and cyclophosphamide, in the isolated perfused rabbit heart model. Initial observations on its cardiotoxicity and cardiac metabolism. Cell Mol Biol (Noisy-le-grand) 43(5):773–782Google Scholar
  34. Kang SH, Chess-Williams R, Anoopkumar-Dukie S, McDermott C (2013) Induction of inflammatory cytokines and alteration of urothelial ATP, acetylcholine and prostaglandin E2 release by doxorubicin. Eur J Pharmacol 700(1–3):102–109. CrossRefPubMedGoogle Scholar
  35. Kehrer JP, Biswal SS (2000) The molecular effects of acrolein. Toxicol Sci 57(1):6–15CrossRefGoogle Scholar
  36. Klinger MB, Dattilio A, Vizzard MA (2007) Expression of cyclooxygenase-2 in urinary bladder in rats with cyclophosphamide-induced cystitis. Am J Physiol Regul Integr Comp Physiol 293(2):R677–R685. CrossRefPubMedGoogle Scholar
  37. Korkmaz A, Topal T, Oter S (2007) Pathophysiological aspects of cyclophosphamide and ifosfamide induced hemorrhagic cystitis; implication of reactive oxygen and nitrogen species as well as PARP activation. Cell Biol Toxicol 23(5):303–312. CrossRefPubMedGoogle Scholar
  38. Kumar V, Chapple CR, Surprenant AM, Chess-Williams R (2007) Enhanced adenosine triphosphate release from the urothelium of patients with painful bladder syndrome: a possible pathophysiological explanation. J Urol 178(4 Pt 1):1533–1536. CrossRefPubMedGoogle Scholar
  39. Kumar V, Chapple CR, Rosario D, Tophill PR, Chess-Williams R (2010) In vitro release of adenosine triphosphate from the urothelium of human bladders with detrusor overactivity, both neurogenic and idiopathic. Eur Urol 57(6):1087–1092. CrossRefPubMedGoogle Scholar
  40. Lawson M, Vasilaras A, De Vries A, Mactaggart P, Nicol D (2008) Urological implications of cyclophosphamide and ifosfamide. Scand J Urol Nephrol 42(4):309–317. CrossRefPubMedGoogle Scholar
  41. Macallister SL, Martin-Brisac N, Lau V, Yang K, O’Brien PJ (2012) Acrolein and chloroacetaldehyde: an examination of the cell and cell-free biomarkers of toxicity. Chem Biol Interact. CrossRefPubMedGoogle Scholar
  42. Macedo FY, Baltazar F, Mourao LC et al (2008) Induction of COX-2 expression by acrolein in the rat model of hemorrhagic cystitis. Exp Toxicol Pathol 59(6):425–430. CrossRefPubMedGoogle Scholar
  43. Macedo FY, Mourao LT, Palheta RC Jr et al (2011) Cyclooxygenase-2 contributes to functional changes seen on experimental hemorrhagic cystitis induced by ifosfamide in rat urinary bladder. Cancer Chemother Pharmacol 67(4):935–943. CrossRefPubMedGoogle Scholar
  44. Mansfield KJ, Hughes JR (2014) Effect of inflammatory mediators on ATP release of human urothelial RT4 cells. Biomed Res Int 2014:182862. CrossRefPubMedPubMedCentralGoogle Scholar
  45. McDermott C, Chess-Williams R, Grant GD et al (2012) Effects of Pseudomonas aeruginosa virulence factor pyocyanin on human urothelial cell function and viability. J Urol 187(3):1087–1093. CrossRefPubMedGoogle Scholar
  46. Mohammad MK, Avila D, Zhang J et al (2012) Acrolein cytotoxicity in hepatocytes involves endoplasmic reticulum stress, mitochondrial dysfunction and oxidative stress. Toxicol Appl Pharmacol 265(1):73–82. CrossRefPubMedPubMedCentralGoogle Scholar
  47. Nissim I, Horyn O, Daikhin Y, Luhovyy B, Phillips PC, Yudkoff M (2006) Ifosfamide-induced nephrotoxicity: mechanism and prevention. Cancer Res 66(15):7824–7831. CrossRefPubMedGoogle Scholar
  48. Pendyala L, Creaven PJ, Schwartz G et al (2000) Intravenous ifosfamide/mesna is associated with depletion of plasma thiols without depletion of leukocyte glutathione. Clin Cancer Res 6(4):1314–1321PubMedGoogle Scholar
  49. Rong W, Spyer KM, Burnstock G (2002) Activation and sensitisation of low and high threshold afferent fibres mediated by P2X receptors in the mouse urinary bladder. J Physiol 541(Pt 2):591–600CrossRefGoogle Scholar
  50. Sadananda P, Shang F, Liu L, Mansfield KJ, Burcher E (2009) Release of ATP from rat urinary bladder mucosa: role of acid, vanilloids and stretch. Br J Pharmacol 158(7):1655–1662. CrossRefPubMedPubMedCentralGoogle Scholar
  51. Schwerdt G, Gordjani N, Benesic A et al (2006) Chloroacetaldehyde- and acrolein-induced death of human proximal tubule cells. Pediatr Nephrol 21(1):60–67. CrossRefPubMedGoogle Scholar
  52. Sellers D, Chess-Williams R, Michel MC (2018) Modulation of lower urinary tract smooth muscle contraction and relaxation by the urothelium. Naunyn Schmiedebergs Arch Pharmacol 391(7):675–694. CrossRefPubMedGoogle Scholar
  53. Sladek NE (1988) Metabolism of oxazaphosphorines. Pharmacol Ther 37(3):301–355CrossRefGoogle Scholar
  54. Sood C, O’Brien PJ (1993) Molecular mechanisms of chloroacetaldehyde-induced cytotoxicity in isolated rat hepatocytes. Biochem Pharmacol 46(9):1621–1626CrossRefGoogle Scholar
  55. Takamoto S, Sakura N, Namera A, Yashiki M (2004) Monitoring of urinary acrolein concentration in patients receiving cyclophosphamide and ifosphamide. J Chromatogr B Analyt Technol Biomed Life Sci 806(1):59–63. CrossRefPubMedGoogle Scholar
  56. Tanaka I, Nagase K, Tanase K, Aoki Y, Akino H, Yokoyama O (2011) Modulation of stretch evoked adenosine triphosphate release from bladder epithelium by prostaglandin E. J Urol 185(1):341–346. CrossRefPubMedGoogle Scholar
  57. Tanel A, Averill-Bates DA (2007) Inhibition of acrolein-induced apoptosis by the antioxidant N-acetylcysteine. J Pharmacol Exp Ther 321(1):73–83. CrossRefPubMedGoogle Scholar
  58. Ventura P, Panini R, Abbati G, Marchetti G, Salvioli G (2003) Urinary and plasma homocysteine and cysteine levels during prolonged oral N-acetylcysteine therapy. Pharmacology 68(2):105–114. CrossRefPubMedGoogle Scholar
  59. Yokoyama O, Tanaka I, Kusukawa N et al (2011) Antimuscarinics suppress adenosine triphosphate and prostaglandin E2 release from urothelium with potential improvement in detrusor overactivity in rats with cerebral infarction. J Urol 185(6):2392–2397. CrossRefPubMedGoogle Scholar
  60. Yoshida M, Inadome A, Maeda Y et al (2006) Non-neuronal cholinergic system in human bladder urothelium. Urology 67(2):425–430. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Centre for Urology Research, Faculty of Health Sciences and MedicineBond UniversityRobinaAustralia

Personalised recommendations