Protective effects of dietary antioxidants on proton total-body irradiation-mediated hematopoietic cell and animal survival

Abstract

Abstract Dietary antioxidants have radioprotective effects after gamma-radiation exposure that limit hematopoietic cell depletion and improve animal survival. The purpose of this study was to determine whether a dietary supplement consisting of l-selenomethionine, vitamin C, vitamin E succinate, alpha-lipoic acid and N-acetyl cysteine could improve survival of mice after proton total-body irradiation (TBI). Antioxidants significantly increased 30-day survival of mice only when given after irradiation at a dose less than the calculated LD(50/30); for these data, the dose-modifying factor (DMF) was 1.6. Pretreatment of animals with antioxidants resulted in significantly higher serum total white blood cell, polymorphonuclear cell and lymphocyte cell counts at 4 h after 1 Gy but not 7.2 Gy proton TBI. Antioxidants significantly modulated plasma levels of the hematopoietic cytokines Flt-3L and TGFbeta1 and increased bone marrow cell counts and spleen mass after TBI. Maintenance of the antioxidant diet resulted in improved recovery of peripheral leukocytes and platelets after sublethal and potentially lethal TBI. Taken together, oral supplementation with antioxidants appears to be an effective approach for radioprotection of hematopoietic cells and improvement of animal survival after proton TBI.

FIG. 1

Effect of antioxidants on mouse survival after TBI. Panel A: Male ICR mice were fed the control AIN-93G diet (n = 15) or the AIN-93G diet supplemented with antioxidants (AO, n = 15) for 7 days prior to 5.9 Gy TBI. The animals were maintained on their respective diets and observed for 30 days after TBI. Panel B: Survival of animals fed the control diet was compared to that of animals given the antioxidant diet 2 h after 5.9 Gy TBI and maintained on this diet for the remaining 30 days (C → AO, n = 15). Panels C and D: Survival of mice fed the control diet (n = 15) or the antioxidant-supplemented diet for 7 days prior to (AO, n = 15) or 2 h after (C → AO, n = 15) 6.8 Gy TBI. Panels E and F: Survival of mice fed the control diet (n = 15) or the antioxidant-supplemented diet for 7 days prior to (AO, n = 15) or 2 h after (C → AO, n = 15) 7.2 Gy TBI.

FIG. 2

Calculation of LD_50/30 for 1 GeV/nucleon protons in male ICR mice (15 per dose) fed the control AIN-93G diet for 7 days prior to TBI. The 30-day survival after TBI ranging from non-lethal (100% survival, 5.5 Gy) to universally lethal (0% survival, 7.2 Gy) was plotted as a function of radiation dose. Linear regression was used to calculate the dose equivalent of 50% 30-day animal survival.

FIG. 3

Effect of prophylactic antioxidant dietary supplementation on peripheral leukocyte counts after low- and high-dose TBI. Male ICR mice were fed the control AIN-93G or the control diet supplemented with antioxidants (AO) for 7 days prior to 1 Gy or 7.2 Gy TBI and were killed at 4 or 24 h after TBI. Panel A: Total white blood cell (WBC) counts, 0 Gy control and 0 Gy AO (n = 4), 1 Gy control 4 h (n = 13), 1 Gy AO 4 h (n = 12), 1 Gy control 24 h (n = 7), 1 Gy AO 24 h (n = 7), 7.2 Gy control 4 h (n = 6), 7.2 Gy AO 4 h (n = 4), 7.2 Gy control 24 h (n = 7), and 7.2 Gy AO 24 h (n = 7). Panel B: PMN cell counts, 0 Gy control and 0 Gy AO (n = 6), 1 Gy control 4 h (n = 13), 1 Gy AO 4 h (n = 9), 1 Gy control 24 h and 1 Gy AO 24 h (n = 5), 7.2 Gy control 4 h (n = 4), 7.2 Gy AO 4 h (n = 4), 7.2 Gy control 24 h (n = 6), and 7.2 Gy AO 24 h (n = 6). Panel C: Lymphocyte counts, 0 Gy control and 0 Gy AO (n = 3–4), 1 Gy control 4 h (n = 10), 1 Gy AO 4 h (n = 10), 1 Gy control 24 h (n = 7), 1 Gy AO 24 h (n = 7), 7.2 Gy control 4 h (n = 5), 7.2 Gy AO 4 h (n = 5), 7.2 Gy control 24 h (n = 8), and 7.2 Gy AO 24 h (n = 7). Each bar represents mean ± SD.

FIG. 4

Effect of dietary antioxidant (AO) supplementation on bone marrow cell depletion and spleen mass 24 h after TBI. Male ICR mice were fed the control AIN-93G or the control diet supplemented with antioxidants (AO) for 7 days prior to 1 Gy or 7.2 Gy TBI and were killed at 24 h after TBI. Panel A: Twenty-four hours after TBI, animals were killed, both femurs and tibiae were flushed with PBS, and cell counts were determined with a Coulter Counter. Each group represents n = 5–7. Panel B: Twenty-four hours after TBI, animals were killed, and the spleens were harvested, defatted and weighed. Each group represents n = 7. Each bar represents mean ± SD.

FIG. 5

Effect of prophylactic dietary antioxidant supplementation on plasma levels of Flt-3L and TGFβ1 after TBI. Male ICR mice were fed the control AIN-93G diet or the control diet supplemented with antioxidant (AO) for 7 days prior to 1 Gy or 7.2 days prior to 1 Gy or 7.2 Gy TBI and were killed 4 and 24 H after TBI; plasma was separated from peripheral blood and stored at −70°C until further analysis. Panel A: Flt-3L: 0 Gy control and AO (n = 5–7), 1 Gy control and AO 4 h (n = 6–8), 1 Gy control and AO 24 h (n = 6–8), 7.2 Gy control and AO 4 h (n = 8), 7. 2 Gy control and AO 24 h (n = 4). Data are means ± SD. ***P = 0.00008 between 1 Gy control and 1 Gy AO at 4 h. *P = 0.018 between 1 Gy control and 1 Gy AO at 24 h. Panel B: TGFβ1: 0 Gy control and AO (n = 6–8), 1 Gy control and AO 4 h (n = 6–8), 1 Gy control and AO 24 h (n = 6), 7.2 Gy control and AO 4 h (n = 7), 7. 2 Gy control and AO 24 h (n = 3). Data are means ± SD with significant difference accepted as P < 0.05 by Student's t test. **P = 0.0013 between 0 Gy control and 1 Gy control at 4 h. **P = 0.0074 between 0 Gy control and 7.2 Gy control at 4 h. *P = 0.031 between 0 Gy control and 1 Gy AO at 24 h. Data are means ± SD.