In summary, DP completely prevented cancellous bone loss caused by irradiation over this short duration study . Our studies demonstrated that DP diet supplementation was equally effective at preventing the skeletal responses to both low-LET gamma radiation, at a dose equivalent to a single fraction of radiotherapy, or combined proton and HZE ions, simulating space radiation. Therefore DP or its components may provide effective interventions for loss of structural integrity caused by radiotherapy or unavoidable exposure to space radiation incurred over long duration spaceflight.Animals. Male C57BL/6J mice experiments at 16 weeks of age were randomized by weight, individually housed, and assigned to groups . Food and water were made available ad libitum and mice were housed on a 12 hours light/dark cycle. Body weights and food consumption were measured throughout the experiments to monitor animal health . The NASA Ames Research Center and the Brookhaven National Laboratory Institutional Animal Care and Use Committee approved all procedures, and studies were conducted in accordance to the IACUC health and ethical standards. Diets. Diet compositions are shown in Fig. 7. The control diets included the following: Control Diet 1 was LabDiet 5001; Control Diet 2 was purified AIN93G and was used as a control for the AOX-supplemented diet of A. Kennedy and colleagues. Control Diet 3 was AIN93M and served as the control for the DP-supplemented diet. The custom antioxidant diet , was prepared by a commercial vendor based on a previously reported diet composition,plastic flower pots with the base AIN93G diet supplemented with five antioxidants: ascorbic acid , N-acetyl cysteine , L-selenomethionine , dihydrolipoic acid , and vitamin E . All antioxidants were obtained from Sigma .

The DP diet was composed of 25% by weight powdered dried plum and was prepared by Teklad as reported by Halloran et al.40. Analysis of custom diets. The candidate interventions were selected in part on the basis of their antioxidant properties. Antioxidant capacity of the custom diets was measured using a Total Antioxidant Capacity measurement assay. In this assay, levels of Trolox equivalents are positively correlated with antioxidant content. DP and DHLA diets had significantly higher antioxidant capacities compared to their respective controls. Specifically, Control diet 1 contained 0.05mM Trolox equivalent per mg of protein. Control diets 2 and 3, CD2 ; and CD3 had lower TAC compared to CD1, 56% and 41% respectively. Both the antioxidant cocktail and dried DP diets had 84% and 74% increased TAC compared to CD1, and their respective controls at 323% and 196% TAC. The AOX and DP diets had comparable TAC’s . Feeding and injection protocols. Mice were separated into groups and fed various diets or injected with DHLA , Ibuprofen . Mice were pre-fed 7 days , 17 days or 21 days with the diets , AOX or DP prior to total body irradiation . The differences in pre-feeding periods were based on previous published findings showing effective prevention of radio damage by AOX diet or bone loss prevention by DP. Feeding with the corresponding diets was continued until euthanasia. For the injection protocols, DHLA or Ibuprofen was delivered via subcutaneous injection twice a day, starting one day prior to TBI as previously reported. In all experiments, mice were irradiated at 16 weeks of age and tissues harvested 1 day later for the gene expression analysis, and 11 days later for the microCT analysis .Radiation exposure. Conscious mice were exposed at 16 weeks of age to 2 Gy Gamma or with 1 Gy of protons and 56Fe ions delivered sequentially to simulate space radiation. The sequential ion exposure was comprised of an initial exposure to 25cGy of 1 H , then 50cGy of 56Fe , and, finally, 25cGy of 1 H; for a total dose of 1Gy, and was performed at the NASA Space Radiation Laboratory at Brookhaven National Laboratory , NY. This exposure regimen was designed to simulate space radiation combining both low-LET and high-LET particles.

Controls were handled identically to the irradiated animals with the exception of exposure to the radiation source, and are referred to as ‘sham-irradiated’. Gene expression. Flushed bone marrow cells from the femora and tibiae were pelleted and lysed in RLT buffer with 1% beta-mercaptoethanol and stored at −80C. Total RNA was extracted using QIAshredder, and RNeasy mini kit . For each sample, RNA was treated with RNase-free DNase Set . RNA quantity was determined using a spectrophotometer and quality confirmed by 2100 Bioanalyzer . Equal amounts of RNA were reversed transcribed, followed by qPCR using GoTaq® RT-qPCR System . Taqman gene expression assays were used : nuclear factor, erythroid 2-like 2 , receptor activator of nuclear factor kappa-B ligand , osteoprotegerin , tumor necrosis factor alpha and monocyte chemotactic protein-1 . The relative gene expression was quantified using the comparative threshold cycle method with normalization to expression levels of glyceraldehyde 3-phosphate dehydrogenase . The reactions were performed in a 7300 RT-PCR System as described previously. Microcomputed tomography . Tibiae were dissected, cut distal to the TFJ to allow PFA infiltration and fixed for 24 hours at 4 °C, followed by storage in 70% Ethanol. The bones were transferred to phosphate-buffered saline and then the proximal metaphysis scanned and analyzed as previously described with a 6.7 μm/voxel resolution using a SkyScan 1174 microCT scanner . For cancellous bone, a 1.0mm thick region located 0.24mm distal to the proximal growth plate of the tibia was selected and semi-autonomously contoured to include cancellous tissue. To assess cancellous bone loss, the bone volume to total volume fraction , trabecular thickness , trabecular number , and trabecular separation were calculated and reported following conventional guidelines. To measure possible changes in cortical features that contribute to whole bone mechanical properties, bones were scanned at 14.6μm/voxel resolution starting at midshaft 2mm proximal to the tibia–fibula junction over a 0.3mm height. Parameters reported include cortical bone cross-sectional area , bone periosteal perimeter , cortical thickness and mean polar moment of inertia .

Tissue mineral density was calculated using the linear attenuation coefficient and calibrated phantoms for diaphyseal cortical bone. Statistics A one-way or two-way analysis of variance was performed as indicated in the legends, with treatment and irradiation as main effects. Where the main effect P< 0.05 by 1-factor ANOVA, or interaction effects by 2-factor ANOVA, differences between groups were analyzed by Dunnett’s post-hoc test comparing experimental groups to the non-irradiated controls, or all pairs Tukey-Kramer . All data are presented as mean and standard deviations.To be labeled as a fruit juice, the US Food and Drug Administration mandates that a product be 100% fruit juice. For juices reconstituted from concentrate, the label must state that the product is reconstituted from concentrate. Any beverage that is less than 100% fruit juice must list the percentage of the product that is fruit juice, and the beverage must include a descriptive term, such as “drink,” “beverage,”or “cocktail.” In general, juice drinks contain between 10% and 99% juice and added sweeteners, flavors, and sometimes fortifiers, such as vitamin C or calcium. These ingredients must be listed on the label according to Food and Drug Administration regulations.Water is the predominant component of fruit juice. Carbohydrates, including sucrose, fructose, glucose, and sorbitol,plastic garden container are the next most prevalent nutrients in juice. The carbohydrate concentration varies from 11 g % to >16 g % . Human milk and standard infant formulas have a carbohydrate concentration of 7 g %. Juice contains a small amount of protein and minerals. Some juices have naturally occurring high contents of potassium, vitamin A, and vitamin C. In addition, some juices and juice drinks are fortified with vitamin C. Juices fortified with calcium have approximately the same calcium content as milk but lack some other nutrients present in milk, such as magnesium and a substantial amount of protein. Many such calcium-fortified juices also are fortified with vitamin D. The vitamin C and flavonoids in juice may have beneficial long-term health effects, such as decreasing the risk of cancer and heart disease.‍ Drinks that contain ascorbic acid consumed simultaneously with food can increase iron absorption by twofold,which may be important for children who consume diets with low iron bio-availability. Juice contains no fat or cholesterol, and unless the pulp is included, it contains no fiber. The fluoride concentration of juice and juice drinks varies. One study found that fluoride ion concentrations in juice ranged from 0.02 to 2.8 ppm.‍The fluoride content of concentrated juice varies with the fluoride content of the water used to reconstitute the juice. Some manufacturers specifically produce juice for infants. These juices do not contain sulfites or added sugars and are more expensive than regular fruit juice. Other forms of fruit juices are frequently consumed. Up to one third of adolescents consume sport drinks, and approximately 10% to 15% consume energy drinks.Pediatricians should inquire about the use of these products as they assess the nutritional intake of their patients.Juices from many fruit contain flavonoids , which can decrease the activity of several enzymes and transport proteins important in drug disposition.‍

Although the ingestion of grapefruit juice has been shown to reduce the activity of intestinal cytochrome P450 3A4 and produce potential drug-nutrient interactions of drugs that are CYP3A4 substrates , recent evidence suggests that grapefruit juice can also inhibit organic acid transporter activity.In addition to grapefruit juice, flavonoids present in oranges and apples have also been shown to reduce the activity of the organic acid transporter OATP2B1.Although the grapefruit juice–CYP3A4 substrate interaction and the potential for producing significant nutrient-drug interactions is the most well characterized, it should be noted that, in addition to inhibiting CYP3A4 activity, cranberry, pomegranate, and blueberry juice can inhibit the activity of CYP2C a cytochrome P450 isoform that catalyzes the bio-transformation of therapeutic drugs such as ibuprofen, flurbiprofen, warfarin, phenytoin, fluvastatin, and amitriptyline. The clinical significance of any of the aforementioned juice-drug interactions is extremely difficult to predict on the basis of a history of coingestion. Substantial variability in the duration and magnitude of resultant interactions is a function of multiple factors, including the following: constitutive expression of the effected enzyme or transport protein, significant genetic polymorphism in the enzyme or transporter, the relative flavonoid composition and potency among different juices, and the amount of juice ingested and its duration of ingestion .In evaluating the potential juice-drug interactions, the coadministration of fruit juice and a drug for which metabolism or transport could be affected by a flavonoid should not be considered immediately as a contraindication for treatment. The amount and type of juice being ingested,9specific information characterizing a given interaction, and whether the drug being taken has a low or high therapeutic index must be considered in the evaluation of a potential interaction. Consultation between the physician and pharmacist can be beneficial in considering the potential clinical significance of a juice-drug interaction.The 4 major sugars in juice are sucrose, glucose, fructose, and sorbitol. Sucrose is a disaccharide that is hydrolyzed into 2 component monosaccharides, glucose and fructose, by sucrase present in the small bowel epithelium. Glucose is then absorbed rapidly via an active-carrier–mediated process in the brush border of the small bowel. Fructose is absorbed by a facilitated transport mechanism via a carrier but not against a concentration gradient. In addition, fructose may be absorbed by a disaccharidase-related transport system, because the absorption of fructose is more efficient in the presence of glucose, with maximal absorption occurring when fructose and glucose are present in equimolar concentrations.‍Clinical studies have shown this process, with more apparent malabsorption when fructose concentration exceeds that of glucose than when the 2 sugars are present in equal concentrations .‍However, when provided in appropriate amounts , these different juices are absorbed equally as well.Sorbitol, found in small amounts in pears, apples, cherries, apricots, and plums and in sugar-free foods and some liquid medications, is absorbed via passive diffusion at slow rates, resulting in much of the ingested sorbitol being unabsorbed.Carbohydrate that is not absorbed in the small intestine is fermented by bacteria in the colon. This bacterial fermentation results in the production of hydrogen, carbon dioxide, methane, and the shortchain fatty acids acetic, propionic, and butyric. Some of these gases and fatty acids are reabsorbed through the colonic epithelium, and in this way, a portion of the malabsorbed carbohydrate can be scavenged.‍Nonabsorbed carbohydrate presents an osmotic load to the gastrointestinal tract, which causes diarrhea.‍ 19