15. In Vivo Radioprotection of Stable Free Radical Nitoxides, Carbamoyl- and Methoxycarbonyl-PROXYL against Whole Body X-Ray Irradiation of Mice

Kazunori Anzai, Masako Furuse, Hiroshi Ishihara and Nobuo Ikota

Keywords: radioprotection, stable nitroxide radical, X-ray irradiation, mice

Radiation induced biological damages are thought to be initiated and propagated via free radical reactions. Therefore, antioxidants are possible candidates for the radiation protection agents. Among these antioxidants, stable nitroxide radicals are interesting because they have superoxide dismutase-like activity. In addition, the radiation protection activity of stable nitroxide radicals has been reported. Methoxycarbonyl-PROXYL (MC-PROXYL) is one of the stable nitroxide radicals. It has a unique property; it is moderately lipophilic and blood-brain-barrier permeable. Previously, we have demonstrated the distribution of MC-PROXYL to mouse brain using autoradiography and in vivo ESR. In the present study, we examined in vivo radiation protection activity of MC-PROXYL against whole body X-ray irradiation of mice in comparison with carbamoyl-PROXYL, a similar but more hydrophilic stable nitroxide radical.

Mice (C3H, male, 10 weeks old) were placed in a chamber after the i.p. administration of MC-PROXYL (450 mg/kg body wt.) and were X-ray irradiated (8.0 Gy, 0.6 Gy/min) at 5 min after the administration of MC-PROXYL. MC-PROXYL increased the survival rate from 0% to 40-50%, showing radiation protection activity. The survival rate was dependent on the timing of the administration and the dose of MC-PROXYL. The condition of i.p. administration at 5 min before the X-irradiation and 450 mg/kg body wt. was the best. A higher dose of MC-PROXYL than 450 mg/kg body wt. caused acute toxicity. MC-PROXYL (450 mg/kg body wt.) increased the LD_50/30 from 6.7 Gy to 8.0 Gy, yielding the dose reduction factor (DRF) of 1.2. This radiation protection activity of MC-PROXYL was larger than that of carbamoyl-PROXYL (DRF = 1.1). The distribution of carbamoyl- and MC-PROXYL to the bone marrow was measured by using ESR. At 5 min after the i.p. administration of carbamoyl- or MC-PROXYL, the mice were killed and bone marrow cells were collected from the thigh bones. ESR spectra of the cell suspensions after oxidation by 1 mM K₃Fe(CN)₆ showed that the distribution of MC-PROXYL in the bone marrow was similar for MC-PROXYL and carbamoyl-PROXYL. Therefore, the difference in radioprotection was not due to any difference in distribution of the chemicals to the bone marrow. A combined administration of MC-PROXYL with heat-killed Lactobacillus casei preparation (LBC) further increased the radiation protection activity. Since the concentration of LBC used in this experiment was in the range showing saturation effect, this finding suggested that the radio-protection mechanism of MC-PROXYL was different from that of LBC.