Lack of a time-dependent effect of melatonin on radiation-induced apoptosis in cultured rat lymphocytes Erkan Yurtcu a , Yildiz Guney b , Mehmet Ali Ergun c, * , H. Zafer Guney d , Canan Uluoglu d , Ayse Hicsonmez b , Berna Yucel d , Gul Ozbey d , Hakan Zengil d a Baskent University Faculty of Medicine, Department of Medical Biology and Genetics, Ankara, Turkey b Ankara University Faculty of Medicine, Department of Radiation Oncology, Ankara, Turkey c Gazi University Faculty of Medicine, Department of Medical Genetics, Besevler, 06500 Ankara, Turkey d Gazi University Faculty of Medicine, Department of Pharmacology, Ankara, Turkey Received 17 October 2006; revised 30 December 2006; accepted 21 March 2007 Abstract Ionizing radiation is widely used for the treatment of solid tumors and it is thought to act by directly targeting tumor clonogens, also known as stem cells. Apoptosis is a genetically programmed mechanism of cell death often characterized by internucleosomal DNA cleavage. Although it has been previously shown that lymphocytes readily undergo apoptosis in patients receiving anticancer drugs or treatment with ionizing ra- diation, this is the first study to investigate the influence of radiotherapy and melatonin on apoptosis in rat lymphocytes at two different times of the day. Melatonin, a free radical scavenger, is an endogenous neurohormone predominantly synthesized in and secreted by the pineal gland. It has been shown that melatonin inhibits apoptosis in normal cells but it increases the rate of apoptosis in various cancer cells. Therefore, in the present study, the effect of melatonin on apoptosis in cultured lymphocytes was studied after total body irradiation (TBI) was given to rats in the morning (1 HALO) or evening (13 HALO) with morphological and DNA fragmentation analysis. Two-way analysis of variance (ANOVA) re- vealed that radiation increased the rate of apoptosis in rat lymphocytes after TBI, and melatonin treatment did not reduce the rate of apoptosis after TBI at either time point. We conclude that the lack of an effect of melatonin on the apoptosis rate in rat lymphocytes might be due to the dose-dependent effect of melatonin, the time course of apoptosis investigated, or the cell type in which apoptosis was examined. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Apoptosis; Total body irradiation; Melatonin; Time dependent; Rat; Lymphocyte 1. Introduction Ionizing radiation is widely used to treat solid tumors and it is thought to act by directly targeting tumor clonogens, also known as stem cells (Hendry et al., 1994; Trott, 1994). The tissue-damaging effect of ionizing radiation is caused by two different mechanisms. The direct action is the breakdown of several molecules in the cells, while indirectly radiation inter- acts with water molecules, causing an increase in the produc- tion of highly reactive free radicals and causing damage to the subcellular structures. Most anticancer treatments, such as radiation therapy (RT), induce DNA damage and apoptosis in normal cells (Vijayalaxmi et al., 2004). Apoptosis is a genetically programmed mechanism of cell death often characterized by internucleosomal DNA cleavage (Szondy, 1997). Since cell death by apoptosis occurs through complex interactions between numerous molecular compo- nents, cells may fail to die when stimulated because of molec- ular abnormalities in the apoptosis pathway or in its control mechanisms. The biochemical hallmark of apoptosis is endo- nuclease-mediated cleavage of internucleosomal DNA linker sections, which initially generates subchromosomal fragments of 50e300 kbp, so-called DNA ladders (Sun et al., 1999). Ap- optotic cleavage of genomic DNA can be identified as DNA * Corresponding author. Tel.: þ90 312 428 1306; fax: þ90 312 202 4635. E-mail address: ergun@tr.net (M.A. Ergun). 1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.03.030 Cell Biology International 31 (2007) 1144e1149 www.elsevier.com/locate/cellbi