Protein nitration, PARP activation and NAD+ depletion may play a critical role in the pathogenesis of cyclophosphamide-induced hemorrhagic cystitis in the rat

Hemorrhagic cystitis (HC) is a major dose-limiting side effect of cyclophosphamide (CP). The mechanism by which CP induces cystitis is not clear. Recent studies demonstrate that nitric oxide; (peroxynitrite) is involved in bladder damage caused by CP. However, the molecular targets of peroxynitrite are not known. The present study is aimed at investigating whether proteins and DNA are molecular targets of peroxynitrite using a rat model. The experimental rats received a single i.p. injection of 150 mg kg(-1) body weight CP in saline and killed 6 or 16 h later. The control rats received saline. The bladders were used for histological and biochemical analysis. Nitrotyrosine and poly-(ADP-ribose) polymerase (PARP) were localized immunohistochemically as indicators of protein nitration and DNA damage, respectively. Nitrite, malondialdehyde, protein thiol and superoxide dismutase (SOD) activity were assayed in the bladder. Hematuria and urinary bladder edema was observed in the CP-treated rats and histologically, moderate to severe damage to the urinary bladder was observed. The bladders of CP-treated rats stained strongly for nitrotyrosine as well as for PARP. Significant decrease in oxidized NAD levels was observed in the bladders of CP-treated rats 16 h following treatment with CP. Protein thiol was depleted and the activity of the peroxynitrite sensitive enzyme SOD was significantly reduced in the bladders of CP-treated rats. The results of the present study reveal that protein nitration, PARP activation and NAD+ depletion may play a critical role in the pathogenesis of CP-induced hemorrhagic cystitis. Based on the results we propose a mechanism for CP-induced cystitis.