Jeffrey H. Ware

Dietary Antioxidants Protect Hematopoietic Cells and Improve Animal Survival after Total-Body Irradiation

Abstract Wambi, C., Sanzari, J., Wan, X. S., Nuth, M., Davis, J., Ko, Y. H., Sayers, C. M., Baran, M., Ware, J. H. and Kennedy, A. R. Dietary Antioxidants Protect Hematopoietic Cells and Improve Animal Survival after Total-Body Irradiation. Radiat. Res. 169, 384–396 (2008). The purpose of this study was to determine whether a dietary supplement consisting of l-selenomethionine, vitamin C, vitamin E succinate, α-lipoic acid and N-acetyl cysteine could improve the survival of mice after total-body irradiation. Antioxidants significantly increased the 30-day survival of mice after exposure to a potentially lethal dose of X rays when given prior to or after animal irradiation. Pretreatment of animals with antioxidants resulted in significantly higher total white blood cell and neutrophil counts in peripheral blood at 4 and 24 h after 1 Gy and 8 Gy. Antioxidants were effective in preventing peripheral lymphopenia only after low-dose irradiation. Antioxidant supplementation was also associated with increased bone marrow cell counts after irradiation. Supplementation with antioxidants was associated with increased Bcl2 and decreased Bax, caspase 9 and TGF-β1 mRNA expression in the bone marrow after irradiation. Maintenance of the antioxidant diet was associated with improved recovery of the bone marrow after sublethal or potentially lethal irradiation. Taken together, oral supplementation with antioxidants appears to be an effective approach for radioprotection of hematopoietic cells and improvement of animal survival, and modulation of apoptosis is implicated as a mechanism for the radioprotection of the hematopoietic system by antioxidants.

Bowman-Birk inhibitor concentrate reduces colon inflammation in mice with dextran sulfate sodium-induced ulcerative colitis

Bowman-Birk inhibitor concentrate (BBIC) is asoybean extract enriched in the BowmanBirk inhibitor, aprotein protease inhibitor. The Bowman-Birk inhibitorcan inhibit proteases released from inflammation mediating cells and suppress superoxide anionradical secretion from immunocytes. This studyinvestigates the ability of Bowman-Birk inhibitorconcentrate to inhibit colon inflammation in the dextransulfate sodium model of ulcerative colitis, aninflammatory bowel disease. When compared to mice on astandard diet, mice given food supplemented with 0.5%BBIC during and after dextran sulfate sodium treatment showed suppression of three of four scoredhistopathological inflammation criteria (P < 0.01),total histopathological score (P < 0.01), a 15% lowermortality rate (P < 0.01), and a delayed onset of mortality. We conclude that dietaryBowman-Birk inhibitor concentrate can beneficiallyaffect dextran sulfate sodium-treated mice and may beuseful in the treatment of human inflammatory boweldiseases, particularly ulcerative colitis.

Effects of dietary supplements on the space radiation-induced reduction in total antioxidant status in CBA mice.

Abstract Guan, J., Stewart, J., Ware, J. H., Zhou, Z., Donahue, J. J. and Kennedy, A. R. Effects of Dietary Supplements on the Space Radiation-Induced Reduction in Total Antioxidant Status in CBA Mice. Radiat. Res. 165, 373–378 (2006). In the present study, the total antioxidant status was used as a biomarker to evaluate oxidative stress induced by proton, HZE-particle and γ radiation in CBA mice. The results demonstrated that the plasma level of TAS was significantly decreased (P < 0.05) in CBA mice after exposure to a 50-cGy dose of radiation from HZE particles or a 3-Gy dose of radiation from protons or γ rays. Diet supplementation with Bowman-Birk Inhibitor Concentrate (BBIC), l-selenomethionine (l-SeM), or a combination of N-acetyl cysteine, sodium ascorbate, co-enzyme Q10 (CoQ10), α-lipoic acid, l-SeM and vitamin E succinate could partially or completely prevent the reduction in the plasma level of TAS in CBA mice exposed to proton or HZE-particle radiation. The selected antioxidant combination with or without CoQ10 has a comparable protective effect on the γ-radiation-induced drop in TAS in CBA mice. These results indicate that BBIC, l-SeM and the selected antioxidant combinations may serve as countermeasures for space radiation-induced adverse biological effects.

Localization of tissue plasminogen activator mRNA in adult rat brain

The distribution of tissue plasminogen activator (tPA) messenger RNA in rat brain was studied using in situ hybridization with 35S UTP-labeled RNA probes derived from a full-length tPA cDNA. Sense strand controls produced low, even backgrounds, with small elevations in the hippocampus. Full-length antisense probes produced strong signals over cerebral ventricular ependyma (including ependyma of the subcommissural organ), meninges, blood vessels, and Purkinje cell layer of the cerebellum, as well as strong signals over scattered cells throughout the brain. Some of these scattered labeled cells were large with lightly stained nuclei, while others were small with darkly stained nuclei. The large labeled cells, which were probably neurons, constituted 6% and 8% of cells in the brain stem and neocortex, respectively, and 100% of Purkinje cells. The small cells, which were present in all areas of the brain, constituted 3-11% of cells in individual brain areas.

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

Abstract Dietary antioxidants have radioprotective effects after γ-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, α-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 LD50/30; 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 TGFβ1 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.

Effects of Dietary Supplements on Space Radiation-Induced Oxidative Stress in Sprague-Dawley Rats

Abstract Guan, J., Wan, X. S., Zhou, Z., Ware, J., Donahue, J. J., Biaglow, J. E. and Kennedy, A. R. Effects of Dietary Supplements on Space Radiation-Induced Oxidative Stress in Sprague-Dawley Rats. Radiat. Res. 162, 572–579 (2004). Of particular concern for the health of astronauts during space travel is radiation from protons and high-mass, high-atomic-number (Z), and high-energy particles (HZE particles). Space radiation is known to induce oxidative stress in astronauts after extended space flight. In the present study, the total antioxidant status was used as a biomarker to evaluate oxidative stress induced by γ rays, protons and HZE-particle radiation. The results demonstrate that the plasma level of total antioxidants in Sprague-Dawley rats was significantly decreased (P < 0.01) in a dose-dependent manner within 4 h after exposure to γ rays. Exposure to protons and HZE-particle radiation also significantly decreased the serum or plasma level of total antioxidants in the irradiated animals. Diet supplementation with l-selenomethionine alone or a combination of selected antioxidant agents was shown to partially or completely prevent the decrease in the serum or plasma levels of total antioxidants in animals exposed to γ rays, protons or HZE particles. These findings suggest that exposure to space radiation may compromise the capacity of the host antioxidant defense and that this adverse biological effect can be prevented at least partially by dietary supplementation with l-selenomethionine and antioxidants.

Effects of Dietary Antioxidant Supplementation on the Development of Malignant Lymphoma and Other Neoplastic Lesions in Mice Exposed to Proton or Iron-Ion Radiation

Abstract Kennedy, A. R., Davis, J. G., Carlton, W. and Ware, J. H. Effects of Dietary Antioxidant Supplementation on the Development of Malignant Lymphoma and Other Neoplastic Lesions in Mice Exposed to Proton or Iron-Ion Radiation. Radiat. Res. 169, 615–625 (2008). Malignancy is considered to be a particular risk associated with exposure to the types of ionizing radiation encountered during extended space flight. In the present study, two dietary preparations were evaluated for their ability to prevent carcinogenesis in CBA mice exposed to different forms of space radiation: protons and highly energetic heavy particles (HZE particles). One preparation contained a mixture of antioxidant agents. The other contained the soybean-derived Bowman-Birk protease inhibitor (BBI), used in the form of BBI Concentrate (BBIC). The major finding was that there was a reduced risk of developing malignant lymphoma in animals exposed to space radiation and maintained on diets containing the antioxidant formulation or BBIC compared to the irradiated animals maintained on the control diet. In addition, the two different dietary countermeasures also reduced the yields of a variety of different rare tumor types observed in the animals exposed to space radiation. These results suggest that dietary supplements could be useful in the prevention of malignancies and other neoplastic lesions developing from exposure to space radiation.

Standardization of a fluorometric assay for measuring oxidative stress in irradiated cells.

Abstract Wan, X. S., Zhou, Z., Ware, J. H. and Kennedy, A. R. Standardization of a Fluorometric Assay for Measuring Oxidative Stress in Irradiated Cells. Radiat. Res. 163, 232–240 (2005). The present study was undertaken to standardize a dichlorofluorescein (DCF) assay for measurement of radiation-induced oxidation of dichlorofluorescin (DCFH) substrate in MCF-10 cells. This assay was highly sensitive and capable of detecting increased DCFH oxidation in the cells exposed to γ radiation at doses as low as 1.5 cGy with linear dose–response curves. However, the slope of the dose–response curves varied considerably from one experiment to another and was influenced by the fluorescent substrate concentration and cell density. To make the assay reproducible so that results obtained from different experiments could be compared, a series of conversion factors and equations have been established to normalize the data for these variables. The results demonstrate that the DCF assay, as standardized in the present study, is highly reproducible with acceptable assay precision. The normalized results can be compared from one experiment to another even when the experiments were performed using different fluorescent substrate concentrations and/or cell densities. Since changes in DCFH oxidation may be related to changes that are indicative of oxidative stress in cells, this assay can be useful to quantify radiation-induced oxidative stress and evaluate the efficacy of antioxidant agents in protection against radiation-induced oxidative stress.

Gamma-radiation (GR) triggers a unique gene expression profile associated with cell death compared to proton radiation (PR) in mice in vivo

Proton radiation (PR) therapy offers a number of potential advantages over conventional (photon) gamma-radiation (GR) therapy for cancer, due to a more localized delivery of the radiation dose. However, the pathophysiological effects following PR-exposure are less well characterized than those of GR-exposure and the molecular changes associated with the acute apoptotic effects in mice in vivo following PR have not been elucidated. Previous studies have estimated the RBE of protons for various in vivo and in vitro endpoints at between 1.1 and 1.3. We assumed an RBE of 1.1 for the endpoints to be evaluated in these studies. Based on this assumption, ICR mice were treated with wholebody doses of GR (1.1 and 7.0 Gy) and PR (1.0 and 6.4 Gy) that were expected to represent RBE-weighted doses. The bone marrow, thymus, spleen and GI-tract were isolated and processed for histology and immunohistochemistry. The apoptotic responses varied greatly between GR and PR in a tissue- and dose-dependent manner. Surprisingly, cell death in the splenic white pulp was consistently lower in PR-treated animals compared to animals treated with GR. This was in spite of an increased presence of damaged DNA following PR as determined by staining for gamma-H2AX and phospho- ATM. Interestingly, both PR and GR triggered nuclear accumulation of p53 and no significant differences were found in the majority of the known pro-apoptotic p53-target genes in the spleens of treated mice. However, GR uniquely triggered a pro-apoptotic expression profile including expression of the pro-apoptotic, p53- and interferon stimulated target gene bcl-g. In contrast to PR, GR may, in a cell type specific manner, trigger a more diverse non-random stress-response that mediates apoptosis partially independent of the extent of DNA damage.

Treatment with soybean-derived Bowman Birk inhibitor increases serum prostate-specific antigen concentration while suppressing growth of human prostate cancer xenografts in nude mice.

Bowman Birk inhibitor (BBI) is an anticarcinogenic serine protease inhibitor that may inhibit the protease activity of prostate‐specific antigen (PSA) and the growth of human prostate cancer xenografts in nude mice.