Pentoxifylline enhances the radioprotective properties of γ-tocotrienol: differential effects on the hematopoietic, gastrointestinal and vascular systems

Abstract

The vitamin E analog γ-tocotrienol (GT3) is a potent radioprotector and mitigator. This study was performed to (a) determine whether the efficacy of GT3 can be enhanced by the addition of the phosphodiesterase inhibitor pentoxifylline (PTX) and (b) to obtain information about the mechanism of action. Mice were injected subcutaneously with vehicle, GT3 [400 mg/kg 24 h before total-body irradiation (TBI)], PTX (200 mg/kg 30 min before TBI), or GT3+PTX before being exposed to 8.5-13 Gy TBI. Overall lethality, survival time and intestinal, hematopoietic and vascular injury were assessed. Cytokine levels in the bone marrow microenvironment were measured, and the requirement for endothelial nitric oxide synthase (eNOS) was studied in eNOS-deficient mice. GT3+PTX significantly improved survival compared to GT3 alone and provided full protection against lethality even after exposure to 12.5 Gy. GT3+PTX improved bone marrow CFUs, spleen colony counts and platelet recovery compared to GT3 alone. GT3 and GT3+PTX increased bone marrow plasma G-CSF levels as well as the availability of IL-1α, IL-6 and IL-9 in the early postirradiation phase. GT3 and GT3+PTX were equally effective in ameliorating intestinal injury and vascular peroxynitrite production. Survival studies in eNOS-deficient mice and appropriate controls revealed that eNOS was not required for protection against lethality after TBI. Combined treatment with GT3 and PTX increased postirradiation survival over that with GT3 alone by a mechanism that may depend on induction of hematopoietic stimuli. GT3+PTX did not reduce GI toxicity or vascular oxidative stress compared to GT3 alone. The radioprotective effect of either drug alone or both drugs in combination does not require the presence of eNOS.

FIG. 1

Kaplan-Meier survival curves for male CD2F1 mice after total-body irradiation. Mice were treated with 10.5 (panel A), 11.5 (panel B) or 12 Gy (panel C). PTX improved survival only in mice treated with 10.5 Gy. GT3 reduced lethality after both 10.5 and 11.5 Gy TBI. Combination treatment prevented TBI-induced mortality up to 12.5 Gy. At 12.5 Gy, the combination therapy was significantly more effective than treatment with PTX or GT3 only. Eight animals per group from a single experiment.

FIG. 2

Kaplan-Meier survival curves for C57BL/6 mice (left panel) and B6.129P2-Nos^Tm1Unc mice (right panel) after 8.5 Gy total-body irradiation. Multivariate analysis revealed a significant effect of treatment with GT3 (P = 0.0001) and PTX (P = 0.002) but not an absolute requirement for eNOS after this dose of radiation. Eight animals per group from a single experiment.

FIG. 3

Panel A: TBI (8.5 Gy) induced a reduction in mucosal surface areas. Administration of a single dose of GT3 24 h before irradiation significantly improved recovery. Combination treatment with GT3 and PTX did not significantly improve postirradiation mucosal surface area compared to treatment with GT3 only (P = 0.08). Treatment with PTX only did not affect mucosal surface area. Four animals per group from a single experiment (means ± SEM). Panel B: Treatment with GT3 and GT3 combined with PTX improved intestinal crypt colony survival (P = 0.0002 and P < 0.0001, respectively). No significant difference was observed between GT3 and GT3 together with PTX. Treatment with PTX only did not improve crypt colony survival. Six animals per group from a single experiment (means ± SEM).

FIG. 4

Panel A: Treatment with PTX, GT3 and GT3 in combination with PTX improved colony-forming units from bone marrow harvested 24 h after 8.5 Gy TBI. Combination therapy was significantly more effective than PTX or GT3 only (P = 0.005 and P = 0.005, respectively). Six mice per group from a single experiment (means ± SEM). Panel B: Treatment with PTX, GT3 and GT3 in combination with PTX improved spleen colony counts 10 days after TBI (8.5 Gy). Combination therapy was significantly more effective than PTX or GT3 only (P = 0.005 and P= 0.005, respectively). Six mice per group from a single experiment (means ± SEM). Panel C: Compared to treatment with GT3 only, combination therapy significantly improved platelet recovery at day 14 postirradiation (8.5 Gy) (P = 0.05). Six mice per group from a single experiment (means ± SEM).

FIG. 5

Panel A: Bone marrow plasma G-CSF levels after 8.5 Gy TBI. GT3, with or without PTX, induced an increase in bone marrow plasma G-CSF at day 0 and day 1 postirradiation. Panel B: Bone marrow plasma IL-1α levels after 8.5 Gy TBI. Treatment with GT3 in combination with PTX induced bone marrow plasma IL-1α levels at day 1 and day 10. Treatment with only GT3 did not increase IL-1α levels at day 1; however, it had an effect on day 10. Panel C: Bone marrow plasma IL-6 levels after 8.5 Gy TBI. IL-6 levels decreased after radiation exposure. GT3+PTX increased the availability of IL-6 at day 1 after TBI. GT3 did not affect IL-6. Panel D: Bone marrow plasma IL-9 levels after 8.5 Gy TBI. GT3+PTX increased the availability of IL-9 at day 1 after TBI. GT3 did not affect IL-9. Panel E: Bone marrow plasma GM-CSF levels after 8.5 Gy TBI. GM-CSF levels were decreased at day 1, 10 and 14 after radiation exposure. None of the treatments affected the postirradiation GM-CSF levels. Six mice per group from a single experiment (means ± SEM). *P < 0.05 compared to vehicle.

FIG. 6

Treatment with GT3 and GT3 combined with PTX reduced postirradiation vascular peroxynitrite production (P = 0.02 and P = 0.02, respectively). No significant difference was observed between GT3 and GT3 together with PTX. Treatment with PTX only did not show an effect. Six mice per group from a single experiment (means ± SEM).