Acrolein, the toxic urinary metabolite of cyclophosphamide, causes physiological changes to bladder function

Introduction: Cyclophosphamide is a commonly administered chemotherapeutic, originally developed for treatment of cancer. In more recent years its effectiveness as an immunotherapeutic agent has been established. However, when cyclophosphamide is metabolised it produces both an active drug and a toxic metabolite, acrolein. Acrolein is excreted in the urine and irritates the bladder wall causing various adverse effects, from urinary frequency, urgency, pain and nocturia, through to haemorrhagic cystitis, which significantly impact on the quality of life of patients. The aim of this study was to investigate the effects of intravesical acrolein on bladder function in isolated whole bladders from mice. It was hypothesised that acrolein would alter the contractility of the bladder and alter nerve-evoked contractions. Materials and methods: Female C57/BL6J mice (12-13 weeks old) were used in this study. Mice were euthanised and bladders were removed and placed under physiological conditions (37°C in aerated Krebs-bicarbonate solution). Whole bladder preparations were performed as previously described by our group. Briefly, the bladders were catheterised with a three-way catheter inserted via the urethra, and intravesical pressure (contractility) was recorded during bladder distension following filling of bladders with saline, or acrolein (100μm) at a rate of 30μl/minute. Spontaneous bladder contractions and compliance and accommodation following distension were measured. Contraction of bladders following stimulation of nerves, and the neurotransmitters involved, was also studied, along with contractions caused by addition of drugs acting at uscarinic, adrenergic and purinergic receptors. AREC: AE150-17, University of Queensland Results: Intravesical acrolein did not affect spontaneous bladder contractions or bladder compliance. The fall in intravesical pressure (accommodation following distension) was less, though not significantly, following acrolein treatment (Figure 1A, p=0.06, one-tailed, unpaired Student’s t-test). Contractility of bladders in response to the muscarinic agonist carbachol was significantly increased in acrolein-infused bladders compared to untreated control bladders (Figure 1B, p<0.05, maximum responses: 31.82±3.12 mmHg vs 23.82±4.00 mmHg respectively, one-tailed, unpaired Student’s t-test). Similarly, bladder contractions evoked by stimulation of nerves at 5Hz were significantly greater (p<0.01) in acrolein-treated bladders, and contractions to the purinergic agonist alpha,betamATP were also significantly increased (p<0.05) (Figure 1C, one-tailed, unpaired Student’s t-test). General detrusor contractility was assessed using potassium chloride and was unchanged by acrolein treatment (p=0.06, one-tailed, unpaired Student’s t-test). Thus, bladder compliance and accommodation were not significantly affected by acrolein treatment, although intravesical acrolein increased contractility to muscarinic and purinergic receptor agonists. This may explain the enhanced contractions following stimulation of nerves in acrolein-treated bladders. Conclusions: Intravesical acrolein enhances contractility of the mouse urinary bladder. This increase in bladder responsiveness may contribute to some of the bladder overactivity and adverse effects experienced in patients following cyclophosphamide treatment.