Chloroacetaldehyde, a metabolite of cyclophosphamide, causes physiological alterations in the urinary bladder.

West E1, Sellers D1, McDermott C1, Chess-Williams R1

Research Type

Pure and Applied Science / Translational

Abstract Category

Pharmacology

Abstract 470
On Demand Pharmacology
Scientific Open Discussion Session 30
On-Demand
Pharmacology Physiotherapy Animal Study Basic Science
1. Centre for Urology Research, Bond University
Presenter
E

Eleanor West

Links

Abstract

Hypothesis / aims of study
Cyclophosphamide (CPO) is a commonly administered chemotherapeutic originally developed to treat a variety of different cancers. Furthermore, CPO is administered as an immunotherapeutic to treat a range of diseases such as rheumatoid arthritis, lupus nephritis, and interstitial lung disease. However, it is known that patients treated with CPO often experience debilitating urotoxic side effects including urinary frequency, nocturia, urgency, and pain extending to hemorrhagic cystitis that has a significant impact on quality of life (1). CPO has also been used, to varying levels of success, to induce and create rodent models of cystitis (2). The mechanism by which CPO causes urotoxic side effects is most commonly attributed to the urinary metabolite acrolein. However, another metabolite of CPO, chloroacetaldehyde (CAA) may also contribute to the side effects experienced in patients. The aim of this study was to investigate the effects of intravesically administered CAA on bladder function, in an ex-vivo whole bladder preparation.
Study design, materials and methods
Female C57BL/6J mice (12-13 weeks) were used in this study. Mice were euthanized, bladders were isolated and placed in 37°C Krebs-bicarbonate solution aerated with carbogen (95% O2 and 5% CO2) gas. Whole bladder preparations were performed using a three-way catheter inserted via the urethra and equipped with a perfusion pump, pressure transducer, and outflow tap (3). The bladder was distended at a rate of 30µL/minute and changes in pressure were visualized using LabChart software (ADInstruments). Bladders were distended with either 0.9% saline (controls) or CAA (100µM in saline) to a pressure of 20mmHg and allowed to accommodate. Spontaneous activity, bladder compliance and accommodation, detrusor contractility, and nerve-evoked contractile responses were measured. Statistical analysis of control versus treated groups was via one-tailed, unpaired Student’s t-test.
Results
Intravesical administration of CAA increased bladder compliance at pressures above 12mmHg (Figure 1A). However,  accommodation of bladders following filling was not affected. The frequency and amplitude of spontaneous contractions during filling were increased in response to CAA, although not significantly compared to controls (P=0.1 and P=0.058 respectively). Additionally, amplitude and frequency of spontaneous contractions during accommodation were significantly increased in CAA-treated bladders compared to controls (Figure 1B, P<0.01; Figure 1C, P<0.05, one-tailed, unpaired Student’s t-test). The muscarinic agonist carbachol produced concentration-dependent increases in intravesical pressure in both control (n=6) and CAA-treated bladders (n=6). However, neither potency nor contractile response was affected. Depolarisation of efferent nerves by electrical field stimulation produced frequency-dependent increases in intravesical pressure, with a significant increase observed only at 1Hz in the CAA=treated group (n=6) when compared to the controls (Figure 2A, n=6, P<0.05, one-tailed Student’s t-test). Electrical field stimulation was also conducted in the absence and presence of various pharmacological agents and data was normalized to the percentage of contraction at 20Hz in the absence of the antagonists (Figure 2B, n=6 for controls and CAA-treated bladders). N-omega-nitro-L-arginine (LNNA), a nitric oxide synthesis antagonist, decreased nerve-evoked contractions in controls bladders by 1.33±3.08% compared to the CAA-treated bladders where contractions were significantly decreased (P<0.05) by 11.21±5.54% (one-tailed, unpaired Student’s t-test). Atropine, a muscarinic acetylcholine receptor antagonist, decreased nerve-evoked contractions by a similar amount in control bladders (13.75±8.374%) and CAA-treated bladders (9.38±12.73%). Desensitization of purinergic receptors using alpha,beta-methylene ATP (aBmATP) caused a similar and significant decrease of nerve-evoked contractions in control bladders (69.52±4.67%, P<0.01) and CAA-treated bladders (60.05±13.2%, P<0.001, one-tailed, unpaired Student’s t-test).
Interpretation of results
CAA-treatment reduced bladder compliance and increased spontaneous activity of bladders during filling. Spontaneous activity during accommodation was also significantly increased following CAA treatment. Detrusor contractility to cholinergic receptor stimulation was unaltered by CAA treatment, and nerve-evoked contractions were affected only at the lowest frequency. CAA-treatment did not atter the contribution of the various neurotransmitters to the contractions.
Concluding message
Intravesical chloroacetaldehyde causes pharmacological and physiological changes to the mouse urinary bladder. This may underlie the potential contribution of chloroacetaldehyde to urotoxic side effects experienced by patients treated with cyclophosphamide.
Figure 1 Fig 1. A| Bladder compliance B| Amplitude of spontaneous contractions during accommodation C| Frequency of spontaneous contractions (measured over 2 minutes) in the absence (n=6) and presence of CAA (100µM, n=6).
Figure 2 Fig 2. A| Nerve-evoked contractile responses to electrical field stimulation and B| Nerve-evoked contractile activity to 20Hz in the presence of LNNA, atropine, aBmATP. In control (n=6) and CAA-treated bladders (n=6).
References
  1. Drake, M.J., Nixon, P.M., & Crew, J.P. (1998). Drug-induced bladder and urinary disorders. Incidence, prevention, and management. Drug Safety. 19(1) 45-55. doi: 10.2165/00002018-199819010-00004
  2. Batista, C.K., Brito, G.A., Souza, M.L., Leitão, B.T., Cunha, F.Q., and Ribeiro, R.A. (2006). A model of hemorrhagic cystitis induced with acrolein in mice. Brazilian Journal of Medical and Biological Research. 39(11) 1475-1481.
  3. West, E.G, Lang, R., Sellers, D., Chess-Williams, R., & McDermott, C. (2018). Ibuprofen Decreases Spontaneous Activity and Enhances Nerve-Evoked Contractions to Minimize Mitomycin C-Induced Bladder Dysfunction. Journal of Pharmacological Experimental Therapies. 366(2). 282-290. doi: 10.1124/jpet.118.248989
Disclosures
Funding This research was supported by an Australian Government Research Training Program Scholarship Clinical Trial No Subjects Animal Species Mouse Ethics Committee University of Queensland AEC
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