Comparison 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

Abstract

We compared three 3-substituted 2,2,5,5-tetramethylpyrrolidine-N-oxyls (PROXYLs): carbamoyl-, methoxycarbonyl-, and hydroxymethyl-PROXYL (CM-, MC-, and HM-PROXYL, respectively) with respect to radioprotection, prevention of DNA damage, and in vivo distribution in mice. The PROXYLs provided protection to C3H mice against lethal X-irradiation (8 Gy) with the following order of magnitude, HM- > CM- ≈ MC-PROXYL. In contrast, radioprotection at the cellular level assessed by the colony formation of leukemia cell line L5178Y showed no difference among them. The degree of protection from X ray-induced oxidation of DNA bases measured by the formation of 8-hydroxydeoxyguanosine in salmon DNA and the cleavage of DNA measured by electrophoresis of plasmid pBR322 DNA did not differ among the PROXYLs. Redox potentials were also similar for each. However, the blood concentration of the PROXYLs injected ip into the mice showed different maximum concentrations (HM- > CM- ≈ MC-PROXYL), although all reached a maximum at around 5–10 min and gradually decreased thereafter. Their concentration in bone marrow showed a similar pattern, suggesting that the difference in in vivo radioprotection among the three PROXYLs is due to the difference in their distribution to bone marrow. In general, the radioprotection provided by stable nitroxides is affected not only by redox potential and reactivity in vitro but also by pharmacokinetics.

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.