Melatonin prevents the increase in hydroxyl radical-spin trap adduct formation caused by the addition of cisplatin in vitro
Introduction
Cisplatin (Cis-diaminedichloroplatinum II) shows high anti-tumor activating against several types of cancer and has been widely used as an agent for cancer chemotherapy. However, high dose of cisplatin produce nephrotoxic side effects, and the dose of cisplatin must therefore often be limited [1]. To reduce cisplatin-induced nephrotoxicity, hydration, hypertonic saline, diuretics, and various other protective agents have been used, but in many cases they did not provide clinically effective protection against nephrotoxicity.
The pathogenesis of the renal damage caused by cisplatin is generally considered to be due to oxidative damage induced by free radical production [2], [3]. Administration of cisplatin causes an increase in lipid peroxides and a decrease in the activity of enzymes that prevent oxidative stress or protect the kidney from such stress. Therefore, administration of antioxidants such as superoxide dismutase [4], glutathione (GSH) and its ester [5], diethyldithiocarbamates (DDTC) [6], selenium, and bismuth subnitrate, a metallothionein inducer before treatment with cisplatin has been used to protect against nephrotoxicity in experimental animals [7], [8]. These treatments have been reported to diminish the increase in lipid peroxidation and the decrease in protective enzyme activities in the kidney that are induced by cisplatin. Masuda et al. [9] found cisplatin-induced generation of active oxygen radicals in a cell-free system. The intracellular concentration of chloride ions has also been reported to strongly influence the formation of cisplatin species that react with DNA [10].
The mammalian pineal secretory product melatonin, 5-methoxy-N-acetyl-tryptamine, is a highly evolutionarily conserved molecule that is present in organisms ranging from algae to humans; it is likely that melatonin and structurally related compounds may be present in all organisms [11]. Melatonin participates in the control of many important physiological functions including seasonal reproduction, the immune system and circadian rhythms [12]. In addition, melatonin is a potent scavenger of the hydroxyl radical and peroxynitrate, suggesting that it may be useful in treating oxygen radical pathophysiology [13]. Recently, Sener et al. [14] and Hara et al. [15] reported that in animal experiments melatonin confers protection against the oxidative damage associated with cisplatin. A hepatic metabolite of melatonin, 6-hydroxymelatonin, is also known to have similar anti-oxidative capacity [16]. However, the mechanism by which melatonin prevents cisplatin-induced nephrotoxicity has not been fully elucidated yet.
The purpose of the present study was to clarify (1) what kind of reactive oxygen species (ROS) are generated by cisplatin, and whether the concentration of chloride ions influences the induction of for ROS production by cisplatin and (2) whether the radicals are scavenged by melatonin or 6-hydroxymelatonin using the procedure of spin trapping of radicals in vitro.
Section snippets
Chemicals
Melatonin, 6-hydroxymelatonin, xanthine oxidase, hypoxanthine, ethylenediaminetetraacetic acid (EDTA) and reduced glutahione were purchased from Sigma Chemical Co. (St. Louis, MO). The spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was obtained from LABOTEC Ltd. (Tokyo, Japan). Cisplatin was kindly supplied from Nippon Kayaku Co., Ltd., (Tokyo, Japan). Other chemicals were purchased from Wako Pure Chemical Industrials, Ltd., (Tokyo, Japan), and all reagents were of the highest quality
Results
The level of the reactive oxygen species (ROS)-DMPO adduct produced by the addition of cisplatin to phosphate buffer at pH 6.8, 7.4 or 8.0 is shown in Fig. 1. The generation of ROS was induced by the addition of cisplatin only in phosphate buffer at pH 7.4. When cisplatin was added to phosphate buffer at pH 7.4 in the presence or absence of chloride ion, changes occurred in the level of ROS-DMPO adduct formed, as shown in Fig. 2. Generation of ROS was observed in the reaction mixture without
Discussion
It has been proven indirectly that ROS production is involved in the renal damage caused by cisplatin. Masuda et al. [9] reported that cisplatin generated superoxide anion (O2−) and hydroxyl radical (OH), as measured by chemiluminescence using a Cypridina luciferin analog, up on interaction with calf thymus DNA in phosphate-buffered saline (PBS) at pH 7.6. They concluded that a single electron released by the cisplatin-DNA interaction and O2 dissolved in PBS is involved in the production of O2−
Acknowledgements
The authors sincerely thank Prof. Y. Akama (Meisei University) for encouragement though this study and thank also Miss Y. Ishi (St. Marianna University of School Medicine) for assistance in ESR measurement.
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