Dried plum diet protects from bone loss caused by ionizing radiation

Abstract

Bone loss caused by ionizing radiation is a potential health concern for radiotherapy patients, radiation workers and astronauts. In animal studies, exposure to ionizing radiation increases oxidative damage in skeletal tissues, and results in an imbalance in bone remodeling initiated by increased bone-resorbing osteoclasts. Therefore, we evaluated various candidate interventions with antioxidant or anti-inflammatory activities (antioxidant cocktail, dihydrolipoic acid, ibuprofen, dried plum) both for their ability to blunt the expression of resorption-related genes in marrow cells after irradiation with either gamma rays (photons, 2 Gy) or simulated space radiation (protons and heavy ions, 1 Gy) and to prevent bone loss. Dried plum was most effective in reducing the expression of genes related to bone resorption (Nfe2l2, Rankl, Mcp1, Opg, TNF-α) and also preventing later cancellous bone decrements caused by irradiation with either photons or heavy ions. Thus, dietary supplementation with DP may prevent the skeletal effects of radiation exposures either in space or on Earth.

Figure 1. Experiment design.

Male mice were assigned to groups (n = 5–10/group) and pre-fed for 7 to 21 days with the various diets (Control diets, CD; or customized diet, AOX and DP), or injected twice a day with DHLA or Ibuprofen starting one day prior to TBI and until tissue harvest. Mice were exposed at 16 wk of age to TBI with 2 Gy Gamma or 1 Gy of dual protons and ⁵⁶Fe. Tissues were harvested 24 hours later for gene expression or 11 days later for microCT analysis.

Figure 2. Effects of candidate interventions on radiation-induced increase of resorption-related genes.

Mice were fed various diets or injected with DHLA or Ibuprofen, then irradiated with ¹³⁷Cs (2 Gy). Dietary interventions included an antioxidant cocktail (AOX) or dried DP (25% by weight) and three separate control diets (Fig. 1 and Methods). After irradiation (24 hr + / − 20 min), mice were euthanized and bone marrow cells were collected for analysis of gene expression by qPCR. Y-axis values indicate relative expression levels of gene of interest normalized to Gapdh using the ΔCt method. Data shown are mean + S.D. (n = 5–6/group) and analyzed by 1-factor ANOVA. *indicates p < 0.05 compared to CD1/sham-irradiated controls by Dunnett’s post hoc test.

Figure 3. DP diet prevented gamma radiation-induced cancellous bone loss.

Mice were fed control diet (CD3) or DP diet, then irradiated with ¹³⁷Cs (2 Gy). After irradiation (11d), mice were euthanized and tibiae collected. Bones were analyzed by microCT for percent bone volume (BV/TV, Panel A), trabecular number (Tb.N, Panel B), trabecular separation (Tb.Sp, Panel C) and trabecular thickness (Tb.Th, Panel D). Representative images of the cancellous bone microarchitecure in 3D reconstructions using the microCT are shown in (panel E). Data shown are mean + S.D. (n = 8/group) and analyzed by 2-factor ANOVA. *indicates p < 0.05 compared to CD3/sham-irradiated controls by Dunnett’s post hoc test.

Figure 4. Effects of DHLA and Ibuprofen on radiation-induced bone loss.

Mice were injected with DHLA, Ibuprofen or Vehicle as described in the methods, then irradiated with ¹³⁷Cs (2 Gy). After irradiation (11d), mice were euthanized and tibiae collected and analyzed by microCT. (BV/TV, Panel A), Tb.N (Panel B), Tb.Sp (Panel C) and Tb.Th (Panel D). Data shown are mean + S.D. (n = 8/group) and analyzed by 1-factor ANOVA. *indicates p < 0.05 compared to CD1/sham-irradiated controls by Dunnett’s post hoc test.

Figure 5. Effects of AOX diet on cancellous bone loss induced by simulated spaceflight radiation.

Mice were fed control diet (CD2) or AOX diet, then irradiated with dual ions (protons, ⁵⁶Fe) to simulate space radiation. After irradiation (11d), mice were euthanized and tibiae collected then analyzed by microCT. (BV/TV, Panel A), Tb.N (Panel B), Tb.Sp (Panel C) and Tb.Th (Panel D). Data shown are mean + S.D. (n = 10). *indicates p < 0.05 compared to CD2, 0 Gy.

Figure 6. DP, but not DHLA, completely prevented bone loss induced by simulated spaceflight radiation.

Mice were separated into groups (n = 8/group), fed selected diets (CD1 or DP) or were injected with DHLA, then were irradiated with dual ions (protons, ⁵⁶Fe) to simulate space radiation. Mice were euthanized 11 days post-IR, and bones analyzed by microCT for percent bone (BV/TV, Panel A), Tb.N (Panel B), Tb.Sp (Panel C) and Tb.Th (Panel D). Data shown are mean + S.D. (n = 8). *indicates p < 0.05 compared to CD1 0 Gy.

Figure 7. Dietary total antioxidant capacity (TAC) and composition.

(Panel A) TAC was measured in the various diets to assess relative antioxidant quality of the custom food sources. CD1 (Purina 5001) was the laboratory’s standard diet, CD2 (AIN93G), was the control for the AOX-supplemented diet and CD3 (AIN-93M) was the control for DP (Fig. 1 and methods). Data shown are mean + S.D. from 3–4 separate aliquots. *p < 0.05 compared to their respective controls (Panel B). Composition of the diets: Control Diet 1 (CD1) is the LabDiet 5001. The specialized diet CD2 + AOX (i.e. antioxidant cocktail diet) was customized supplementing with 5 antioxidants: ascorbic acid (AA), N-acetyl cysteine (NAC), L-selenomethionine, lipoic acid (DHLA), and vitamin E (See Methods). The customized dried plum (DP) diet is the AIN93M diet with addition of 25% by weight of dried plum powder.