ORIGINAL ARTICLE Melatonin prevents the development of hyperplastic urothelium induced by repeated doses of cyclophosphamide Daša Zupančič & Gaj Vidmar & Kristijan Jezernik Received: 16 October 2008 / Revised: 14 February 2009 / Accepted: 19 March 2009 / Published online: 21 April 2009 # Springer-Verlag 2009 Abstract Repeated cyclophosphamide (CP) chemotherapy increases the risk of developing bladder cancer, which could be due to the extremely rapid proliferation of urothelial cells observed in hyperplastic urothelium induced by CP treatment. We investigated the effect of melatonin on the development of urothelial hyperplasia induced by repeated CP treatment. Male ICR mice were injected with CP (150 mg/kg) or melatonin (10 mg/kg) with CP once a week for 3, 4 and 5 weeks. Transmission and scanning electron microscopy, immunohistochemistry and Western blot analysis were used to study the ultrastructure, apoptosis, proliferation and differentiation of urothelial cells. Repeated doses of CP caused the development of hyperplastic urothelium with up to ten cell layers and increased proliferation and apoptotic indices regarding Ki-67 and active caspase-3 immunohistochemistry, respec- tively. Scanning electron microscopy observations, cytoker- atin and asymmetrical unit membrane immunohistochemistry and Western blot analysis showed a lower differentiation state of superficial urothelial cells. Melatonin co-treatment prevented the development of hyperplastic urothelium, statistically significantly decreased proliferation and apo- ptotic indices after four and five doses of CP and caused higher differentiation state of superficial urothelial cells. Keywords Urothelium . Cyclophosphamide . Hyperplasia . Melatonin Introduction The three-layered mammalian urinary bladder urothelium is a normally very stable and highly differentiated epithelium [1]. Nevertheless, it shows a great capacity for regeneration after a mechanically or chemically induced damage [1–3]. Urothelial regeneration usually gives rise to hyperplasia and can contribute to a wide range of neoplasms [2]. Hicks and Chowaniec [2] and our unpublished data showed that repeated intraperitoneal doses of cyclophosphamide (CP) cause persistent urothelial hyperplasia. Cohen et al. have developed a model of carcinogenesis which incorporates several basic principles: cancer results from genetic errors within normal cells; carcinogenesis is a multi-stage process, requiring more than one genetic event; and a chance of a genetic event occurring per cell division is a finite quantity and, therefore, that increased cell division increases the chance for a genetic event [4]. Based on this model, they then postulate that changes which lead to increased cell proliferation in the urothelium should be carefully evaluat- ed as a possible contributing factor to carcinogenesis [5–7]. CP has long been known to produce severe hyperplasia following cytotoxic damage to the urothelium [8, 9]. However, it is also a weak complete carcinogen and after prolonged application will produce cancer of the urothe- lium, but its hyperplastic action is probably associated with its late-stage carcinogenic activity rather than with its first- stage initiating activity [10]. In a lifelong experiment of the carcinogen action of CP, low-dose oral application of CP led to the induction of bladder cancer in 30% to 43% of rats [11]. However, in the last few months of the experiments, Virchows Arch (2009) 454:657–666 DOI 10.1007/s00428-009-0765-3 D. Zupančič (*) : K. Jezernik Institute of Cell Biology, Faculty of Medicine, Lipičeva 2, 1000 Ljubljana, Slovenia e-mail: dasa.zupancic@mf.uni-lj.si G. Vidmar Institute for Rehabilitation, Linhartova cesta 51, 1000 Ljubljana, Slovenia