Amelioration of radiation-induced hematopoietic and gastrointestinal damage by Ex-RAD(R) in mice

Abstract

The aim of the present study was to assess recovery from hematopoietic and gastrointestinal damage by Ex-RAD(®), also known as ON01210.Na (4-carboxystyryl-4-chlorobenzylsulfone, sodium salt), after total body radiation. In our previous study, we reported that Ex-RAD, a small-molecule radioprotectant, enhances survival of mice exposed to gamma radiation, and prevents radiation-induced apoptosis as measured by the inhibition of radiation-induced protein 53 (p53) expression in cultured cells. We have expanded this study to determine best effective dose, dose-reduction factor (DRF), hematological and gastrointestinal protection, and in vivo inhibition of p53 signaling. A total of 500 mg/kg of Ex-RAD administered at 24 h and 15 min before radiation resulted in a DRF of 1.16. Ex-RAD ameliorated radiation-induced hematopoietic damage as monitored by the accelerated recovery of peripheral blood cells, and protection of granulocyte macrophage colony-forming units (GM-CFU) in bone marrow. Western blot analysis on spleen indicated that Ex-RAD treatment inhibited p53 phosphorylation. Ex-RAD treatment reduces terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay (TUNEL)-positive cells in jejunum compared with vehicle-treated mice after radiation injury. Finally, Ex-RAD preserved intestinal crypt cells compared with the vehicle control at 13 and 14 Gy. The results demonstrated that Ex-RAD ameliorates radiation-induced peripheral blood cell depletion, promotes bone marrow recovery, reduces p53 signaling in spleen and protects intestine from radiation injury.

Fig. 1.

Dose response of Ex-RAD for radioprotection. Kaplan–Meier 30-day survival rates were observed in male C3H/HeN mice (n = 16 per group) treated twice sc, 24 h and 15 min before radiation at 7.5 Gy of cobalt-60 gamma radiation with vehicle (1% Tween-80, 0.1 M KP buffer, 15 mM NaCl, pH 8.2) or Ex-RAD (25–500 mg/kg body weight). Mice that received Ex-RAD dose of 500 mg/kg exhibited a significant increase in survival as compared with vehicle control group and lowest dose of Ex-RAD 25 mg/kg (P < 0.0033, Fisher's exact test). Note: There was a significant difference among the survival curves (P = 0.0014, df = 5, Chi Squared value = 19.67, logrank test).

Fig. 2.

Dose reduction factor (DRF) determination. Mice (n = 16 per group) were treated sc twice, 24 h and 15 min before radiation with vehicle (1% Tween-80, 0.1 M K-P buffer, 15 mM NaCl, pH 8.2) or Ex-RAD, 500 mg/kg body weight. Radiation doses for the vehicle group were 6.25, 7.0, 7.5, 8.0 and 8.5 Gy; doses for Ex-RAD-treated group were 7, 7.5, 8.0, 8.5 and 9.0 Gy. Probit mortality curves were generated and a DRF of 1.16 with 95% confidence interval (CI, 1.13–1.20) was calculated from the ratio of LD_50/30 of Ex-RAD-treated to vehicle-treated mice. Note: The 95% CI is 7.80, 8.22 for Ex-RAD LD_50/30 and 6.66, 7.00 for vehicle LD_50/30, respectively.

Fig. 3.

Ex-RAD pretreatment enhances recovery of peripheral blood cells in irradiated C3H mice. Effect of Ex-RAD on (a) total white blood cells (WBC), (b) absolute neutrophil count (ANC), (c) monocytes (MONO) and (d) platelets (PLT) in mice (n = 10 per group) subjected to total-body gamma radiation with a sublethal dose of 6 Gy (0.6 Gy/min) was measured. Mice were administered sc injection of vehicle (1% Tween-80, 0.1 M KP buffer, 15 mM NaCl, pH 8.2) or Ex-RAD 500 mg/kg body weight, 24 h and 15 min prior to gamma radiation. Day 0 represents the day of irradiation. Data represented are means ± standard error of the mean (SEM) for n = 10 mice for each time point. The marked group (*) indicates significant difference (P < 0.05) between irradiated Ex-RAD compared with irradiated vehicle group by Tukey–Kramer method. Note: Some error bars are very small and hence not visible in the figure.

Fig. 4.

Ex-RAD protects bone marrow granulocyte-macrophage colony-forming units (GM-CFUs). Ex-RAD (500 mg/kg) or vehicle (1% Tween-80, 0.1 M KP buffer, 15 mM NaCl, pH 8.2) was injected sc twice, 24 h and 15 min before exposure to 7 Gy TBI or sham irradiation. A significant (*P < 0.01) increase in colony size was observed in bone marrow cells harvested from Ex-RAD-treated animals on Days 8 and 14 compared with vehicle-treated animals. No differences in GM-CFUs were observed in sham-irradiated, vehicle-treated and drug-treated animals. Each point represents the mean ± SEM of three pools of marrow. Each pool comprised bone marrow from four femurs.

Fig. 5.

Ex-RAD protects mouse jejunum crypt cells from radiation damage post-TBI. Eight-week-old mice were injected sc with vehicle or Ex-RAD before TBI with 13 Gy. Photomicrograph of jejunum sections of mice harvested 3.5 days after irradiation is shown in the top panel. Higher magnification (40×) images of vehicle-treated mice showed shortened, irregular and thickened villi (arrows) with increased number of goblet cells; whereas Ex-RAD-treated mice showed normal villi, restoration of crypt cells (arrow) and normal distribution of goblet cells.

Fig. 6.

Crypts per circumference of jejunum sections from Ex-RAD-treated and vehicle-treated groups 12 h and 3.5 days post radiation (13 and 14 Gy). The number of crypt cells in the Ex-RAD-treated group was significantly higher at both times (*P < 0.001, n = 8) than the vehicle-treated group. Values are means ± SE of the mean of surviving crypts per cross-section.

Fig. 7.

TUNEL staining in the jejunum sections from Ex-RAD-treated and vehicle-treated groups 24 h post-TBI (6 Gy). Mice were sacrificed at the indicated time post-TBI. (a) Cell death in the jejunum sections after IR was assessed by TUNEL staining (magnification 10×), (b) Quantification of TUNEL positive cells in 50 villi/mouse. Values are means ± SEM; n = 3 in each group. * indicates P < 0.0001 compared with vehicle control.

Fig. 8.

Ex-RAD inhibits gamma radiation-induced phospho-p53 (p-p53) activity in spleens of mice. Mice were treated with Ex-RAD (500 mg/kg) before radiation at 6 Gy and spleens were harvested 24 h post-radiation. Spleen homogenates were prepared for total protein for five groups (Naïve, Non-radiated vehicle, Non-radiated Ex-RAD, Irradiated vehicle and Irradiated Ex-RAD; shown as Naïve, NR Veh, NR Ex-RAD, IR Veh and IR Ex-RAD). Endogenous actin expression was shown as control. (a) p53 expression, inhibited by Ex-RAD; (b) p-p53 expression, significantly inhibited by the drug; (c and d) specific p53 and p-p53 bands were quantitated densitometrically, p53 and p-53 levels were normalized to actin levels in each group. *P < 0.05 compared with NR Veh, ** P < 0.05 compared with IR Veh. Error bar indicates SEM for n = 3 independent experiments.