Original ContributionComparison of stable nitroxide, 3-substituted 2,2,5,5-tetramethylpyrrolidine-N-oxyls, with respect to protection from radiation, prevention of DNA damage, and distribution in mice
Introduction
Radiation-induced biological damage is thought to be initiated and propagated via free radical reactions [1], [2]. Therefore, antioxidants potentially provide protection from radiation. Among antioxidants, stable nitroxides are unique and of interest. They have been used as spin probes for ESR experiments for membrane biophysics [3], [4]. Recently, another application for in vivo ESR measurements as redox-sensitive probes has been reported [5], [6]. Interestingly, a metal-independent superoxide dismutase mimetic activity was found in nitroxides [7], [8], [9], [10]. Stable nitroxides protect mammalian cells and cardiomyocytes from oxidative stress [9] and an extensive study of the structure–activity relationship was performed by Krishna et al. [11].
Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), a water-soluble stable nitroxide, was shown to protect cultured mammalian cells exposed to ionizing irradiation [12], [13]. Further, it was shown to provide protection in vivo [14], [15] and was recently tested in a phase one trial [16]. A number of other water-soluble nitroxides also provided protection from radiation in vitro [17]. Among them, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (3-carbamoyl-PROXYL; CM-PROXYL) was reported to offer radioprotection similar in magnitude to Tempol and had little effect on blood pressure [17].
Methoxycarbonyl-PROXYL (MC-PROXYL) and hydroxymethyl-PROXYL (HM-PROXYL) are among the stable nitroxides and 3-substituted pyrrolidine analogs of CM-PROXYL. They have unique properties, being moderately lipophilic and able to permeate the blood–brain barrier [18], [19], [20], [21]. Previously, we have demonstrated the distribution of MC-PROXYL in mouse and rat brain using autoradiography and in vivo ESR [18], [21]. In the present study, we examined in vivo the protective activity of HM- and MC-PROXYL against whole-body X-ray irradiation of mice in comparison with CM-PROXYL, a similar but more hydrophilic stable nitroxide. In addition, cellular protection, protection against DNA damage caused by X-ray irradiation, and distribution to blood and bone marrow were compared to reveal the possible reason for the difference in magnitude of effect in vivo.
Section snippets
Chemicals
CM-PROXYL was a product of Sigma (St. Louis, MO). MC-PROXYL was synthesized from carboxy-PROXYL (Tokyo Kasei, Tokyo, Japan) and diazomethane [21], [22]. HM-PROXYL was also synthesized from carboxy-PROXYL by reduction with LiAlH4 in THF [20], [23]. MC-PROXYL and HM-PROXYL were purified by column chromatography and their purity was checked with TLC and NMR.
Animals
Male C3H mice were obtained from Japan SLC Co. (Hamamatsu, Japan). The mice, received at 8 weeks of age, were housed five per cage and allowed
In vivo radioprotection
CM-, MC-, and HM-PROXYLs were administered ip at 2.25 mmol/kg bw 5 min prior to whole-body irradiation with X-rays (8.0 Gy). As shown in Fig. 1, compared to saline as a control, all PROXYLs were found to have provided radioprotection when survival at 30 days was measured, with HM-PROXYL providing the highest level of protection by a significant margin.
The radioprotection of HM-PROXYL was examined in more detail. Dose-dependent radioprotection was observed as shown in Fig. 2A. A dose of 2.25
Discussion
In the present study, we have shown that all the PROXYLs examined provide protection against radiation both at the cellular level and in vivo. Others have reported that CM-PROXYL provides radioprotection in vivo similar in magnitude to that of Tempol [17]. Here we found that MC-PROXYL does too, and that HM-PROXYL provides far better radioprotection in vivo. The effect of HM-PROXYL is dependent on dose and timing of administration. Maximum activity was observed when it was injected 5 min before
Acknowledgments
We thank Dr. Masahiko Mori and Ms. Hiromi Itsukaichi of this institute for their introduction and help with colony assays. We also thank Ms. Haruko Yakumaru for synthesis of the PROXYLs, Ms. Azusa Matsuyama for measurements of the heart rate, blood pressure, and body temperature of mice, and Dr. Joe Rodrigue for editing the manuscript.
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Present address: Faculty of Pharmaceutical Sciences, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan.