Delayed Captopril Administration Mitigates Hematopoietic Injury in a Murine Model of Total Body Irradiation

Abstract

The increasing potential for accidental radiation exposure from either nuclear accidents or terrorist activities has escalated the need for radiation countermeasure development. We previously showed that a 30-day course of high-dose captopril, an ACE inhibitor, initiated 1-4 h after total body irradiation (TBI), improved Hematopoietic Acute Radiation Syndrome (H-ARS) and increased survival in mice. However, because of the time likely required for the deployment of a stockpiled radiation countermeasure to a radiation mass casualty site, there is a need for therapies that can be administered 24-48 hours after initial exposure. Using C57BL/6 mice exposed to an LD_50-80/30 of ⁶⁰Co TBI (7.75-7.9 Gy, 0.615 Gy/min), we show that low-dose captopril administration, initiated as late as 48 h post-TBI and continued for 14 days, significantly enhanced overall survival similarly to high-dose, rapid administration. Captopril treatment did not affect radiation-induced cell cycle arrest genes or the immediate loss of hematopoietic precursors. Reduced mortality was associated with the recovery of bone marrow cellularity and mature blood cell recovery at 21-30 days post-irradiation. Captopril reduced radiation-induced cytokines EPO, G-CSF, and SAA in the plasma. Finally, delayed captopril administration mitigated brain micro-hemorrhage at 21 days post-irradiation. These data indicate that low dose captopril administered as late as 48 h post-TBI for only two weeks improves survival that is associated with hematopoietic recovery and reduced inflammatory response. These data suggest that captopril may be an ideal countermeasure to mitigate H-ARS following accidental radiation exposure.

Figure 1

Kaplan-Meier Curves of the effects of reduced dosage and delayed administration of captopril on survival from total body irradiation. C57BL/6 mice, 12–14 weeks of age, were exposed to total body ⁶⁰Co irradiation (0.6 Gy/min) or sham irradiated (sham). Irradiated (Rad) mice either received vehicle (Veh) or captopril (Cap), provided in the drinking water. For all irradiated groups, n = 16–20; for the sham irradiated group, n = 8. The percentage of surviving mice are shown. (A) Mice were exposed to 7.5 Gy (LD_50/30) total body irradiation and were treated with either vehicle only (water) or with captopril administration (110 mg/kg/day) beginning at various time points from 1 h to 24 h postirradiation, as indicated, through 30 days postirradiation. (B) Mice were exposed to 7.9 Gy. Mice were untreated or captopril (110 mg/kg/day or 13 mg/kg/day) was administered 4 h through 30 days post-irradiation. (C) Mice were exposed to 7.9 Gy. Mice were untreated or captopril (13 mg/kg/day) was initiated at either 4 h, 24 h, or 48 h post-irradiation through 30 days post-irradiation. (D) Mice were exposed to 7.9 Gy. Mice were untreated or captopril (13 mg/kg/day), administered in the drinking water, was initiated at 48 h post-irradiation through 14, 21 or 30 days post-irradiation. *p < 0.05 compared with radiation + vehicle.

Figure 2

Effect of delayed captopril treatment on mature blood cell loss and recovery after total body irradiation. C57BL/6 mice, 12–14 weeks of age, were exposed to 7.9 Gy total body ⁶⁰Co irradiation (0.6 Gy/min). Mice received vehicle or received captopril (13 mg/kg/day), administered in the drinking water either 48 h through 14 days post-irradiation or 4 h through 30 days post-irradiation. Blood was obtained at 3, 7, 14, 21 and 30 days post-irradiation for analysis and quantification of (A) red blood cells (RBC), (B) reticulocytes, (C) hematocrit (HCT), (D) platelets, and (E) peripheral white blood cells (WBC). Data show means ± standard error of the mean, n = 4–5 mice per group. *p < 0.05 for captopril treatment 4 h–30 d vs radiation + vehicle; ^†p < 0.05 for captopril treatment 48 h–14 d vs radiation + vehicle. ^‡indicates a significant increase for captopril 48 h–14 d vs sham, p < 0.05.

Figure 3

Effect of delayed captopril treatment on bone marrow cellularity following total body irradiation. C57BL/6 mice, 12–14 weeks of age, were exposed to 7.9 Gy total body ⁶⁰Co irradiation (0.6 Gy/min) or sham irradiated (sham). Mice received vehicle (7.9 Gy + vehicle) or received captopril (13 mg/kg/day, 7.9 Gy + Cap), administered in the drinking water either 48 h through 14 days post-irradiation. (A) Sternabrae were obtained from mice at the indicated time points and processed for H&E staining. (B) Bone marrow cellularity was scored by a hematological pathologist blinded to the treatment groups, n = 3–5 per group, except for the 30 day time point for radiation + vehicle, which had only one animal (indicated by^†).

Figure 4

Effect of delayed captopril treatment on senescent-associated gene expression in the bone marrow following total body irradiation. C57BL/6 mice, 12–14 weeks of age, were exposed to 7.9 Gy total body ⁶⁰Co irradiation (0.6 Gy/min) or sham irradiated (sham). Mice received vehicle (7.9 Gy + vehicle) or received captopril (13 mg/kg/day, 7.9 Gy + Cap), administered in the drinking water 48 h – 14 days post-irradiation. Bone marrow was obtained at the indicated times and RT-qPCR was performed to quantify mRNA for the following cell cycle genes: (A) Cdkn2b; (B) Cdkn1a; (C) Cdkn2a. Data show means ± SEM, n = 3–5 per group, except for the 30 day time point for radiation + vehicle, which had only one animal (indicated by ^‡). *Indicates p < 0.05 between radiation + vehicle and sham; ^†indicates p < 0.05 between radiation + captopril and sham.

Figure 5

Effect of delayed captopril administration on cytokine levels in peripheral blood following total body irradiation. C57BL/6 mice, 12–14 weeks of age, were exposed to 7.9 Gy total body ⁶⁰Co irradiation (0.6 Gy/min) or sham irradiated (sham). Mice received vehicle (7.9 Gy + vehicle) or received captopril (13 mg/kg/day, 7.9 Gy + Cap), administered in the drinking water 48 h – 14 days post-irradiation. Serum was obtained at the indicated times, and the following growth factors and cytokines were quantified by MSD or ELISA: (A) EPO; (B) G-CSF; C. SAA1; (D) IL-6. Data show means ± SEM, n = 3–5 per group, except for the 30 day time point for radiation + vehicle, which had only one animal (indicated by ^§). *Indicates p < 0.05 between radiation + vehicle and sham; ^†indicates p < 0.05 between radiation + captopril and sham; ^‡indicates p < 0.05 between radiation + vehicle and radiation + captopril.

Figure 6

Captopril reduces brain hemorrhage following 7.9 Gy total body irradiation at 21 days post-irradiation. C57BL/6 mice, 12–14 weeks of age, were exposed to 7.9 Gy total body ⁶⁰Co irradiation (0.6 Gy/min) or sham irradiated (sham). Mice received vehicle (7.9 Gy + vehicle) or received captopril (13 mg/kg/day, 7.9 Gy + Cap), administered in the drinking water 48 h – 14 days post-irradiation. At 21 days days post-irradiation, brains were obtained after euthanasia and fixed for histology. (A) Representative histological sections of the cerebellum and hippocampus from mice. (B) Brain injury scores, performed by a pathologist blinded to the treatment group. Bar graphs show means ± SEM, n = 3–4 per group