Melatonin ameliorates bladder damage induced by cyclophosphamide in rats Introduction Hemorrhagic cystitis (HC) is a major potential toxicity and dose-limiting side effect of cyclophosphamide (CP) and ifosfamide, a synthetic analog of CP [1]. The incidence of this side effect is related to the dosage and can be as high as 75% in patients receiving a high intravenous dose. The urological side effects vary from transient irritative voiding symptoms to life-threatening HC. The urotoxicity of these nitrogen mustard cytostatics is not based on a direct alkylating activity on the urinary system but rather on the formation of 4-hydroxy metabolites, in particular, renal excretion of acrolein, which is formed from hepatic microsomal enzymatic hydroxylation. However, detoxify- ing acrolein does not prevent HC symptoms completely [2, 3]. Recently, it was shown that increasing nitric oxide (NO) production is responsible for the detrimental effects of CP on urinary bladder [4, 5]. Nitric oxide is a free-radical gas that regulates a number of important physiologic and pathophysiologic processes including vascular tone, polymorphonuclear leukocyte (PMN) adhesion, and inflammation. NO is produced by a family of enzymes called NO synthases (NOS) through enzymatic oxidation of the guanidino group of l-arginine [6]. Constitutive expression of two NOS isoforms is responsible for a low basal level of NO synthesis in neural cells (nNOS) and endothelial cells (eNOS). Induction of the inducible isoform (iNOS) by cytokines has been observed in virtually all cell types including macrophages, fibroblasts, epithelial cells, and results in the production of large amounts of NO. There is evidence that the NO produced by iNOS is toxic, as in animal models, selective iNOS inhibition improves outcome and decreases inflammatory events [7]. This toxicity is probably derived from reactive nitrogen species (RNS), in particular, peroxynitrite (ONOO ) ) overproduction when NO couples with superox- ide (O ÀÁ 2 ), which appears abundantly in the inflammatory area [8]. Moreover, it is clear that, in biological systems the primary source of RNS is NO. The overproduction of reactive oxygen species (ROS) and RNS during inflamma- tion leads to an extensive oxidative stress, cellular injury and apoptosis/necrosis via several mechanisms including peroxidation of membrane lipids, protein denaturation and DNA damage [9]. Thwarting the damage inflicted by free radicals and reactive species is the function of a complex antioxidative defense system. This system includes enzymes such as superoxide dismutase, catalase and glutathione peroxidase and some of the most commonly used and experimentally studied non-enzymatic antioxidants, including melatonin [10, 11]. Moreover, melatonin is known to be a potent iNOS inhibitor [12] and peroxynitrite scavenger [13]. Abstract: Cyclophosphamide (CP), an alkylating antineoplastic agent, has potential urotoxicity including causing hemorrhagic cystitis (HC). HC is now accepted as a non-infectious inflammation and the pathogenesis of HC includes cytokine production which leads to inducible nitric oxide synthase (iNOS) induction. Moreover, overproduction of reactive oxygen species (ROS) during inflammation leads to extensive oxidative stress, cellular injury and apoptosis/necrosis via several mechanisms. Based on these facts, the aim of this study was to evaluate the protective effects of melatonin as an antioxidant, iNOS inhibitor and peroxynitrite scavenger against CP-induced urinary bladder damage. A total of 30 male Sprague–Dawley rats were divided into four groups. Three groups received a single dose of CP (100 mg/kg) intraperitoneally with the same times. Group 2 received CP only, group 3 received 5 mg/kg/day and group 4 received 10 mg/kg/day melatonin before and the day after CP administration. Group 1 served as the control. Increased iNOS induction, bladder malonyldialdehyde (MDA) levels and urinary nitrite–nitrate excretion were encountered in the CP-only group leading to severe cystitis. Melatonin exhibited significant protection against CP-induced cystitis by diminishing bladder oxidative stress and blocking iNOS and peroxynitrite production. Oxidants may have a major role in the pathogenesis of CP-induced cystitis and iNOS is an important mediator leading to peroxynitrite production. Melatonin ameliorates bladder damage induced by CP. Turgut Topal 1 , Yesim Oztas 2 , Ahmet Korkmaz 1 , Serdar Sadir 1 , Sukru Oter 1 , Omer Coskun 3 and Hayati Bilgic 1 1 Department of Physiology, Gulhane Military Medical Academy, Ankara; 2 Department of Biochemistry, Ankara University School of Medicine, Ankara; 3 Department of Medical Histology and Embryology, Karaelmas University School of Medicine, Zonguldak, Turkey Key words: antioxidant, cyclophosphamide, cystitis, melatonin, nitric oxide, peroxynitrite Address reprint request to Ahmet Korkmaz, GATA Fizyoloji AD, 06018 Etlik/Ankara, Turkey. E-mail: korkmaza@gata.edu.tr; fizyocan@ hotmail.com Received August 13, 2004; accepted November 2, 2004. J. Pineal Res. 2005; 38:272–277 Doi:10.1111/j.1600-079X.2004.00202.x Copyright Ó Blackwell Munksgaard, 2005 Journal of Pineal Research 272