X. Steven Wan

Adaptation of the Dichlorofluorescein Assay for Detection of Radiation-Induced Oxidative Stress in Cultured Cells

Abstract Wan, X. S., Zhou, Z. and Kennedy, A. R. Adaptation of the Dichlorofluorescein Assay for Detection of Radiation-Induced Oxidative Stress in Cultured Cells. Radiat. Res. 160, 622–630, (2003). The oxidation of 2′7′-dichlorofluorescin (DCFH) to 2′7′-dichlorofluorescein (DCF), a fluorescent DCFH oxidation product, is a highly sensitive indicator that is used to measure oxidative stress in cells. In the present study, a DCF assay has been adapted to quantify oxidative stress in human breast epithelial cell cultures after exposure to γ rays. The results demonstrate that the sensitivity and specificity of the DCF assay is strongly influenced by the timing of DCFH diacetate (DCFH-DA) substrate loading in relation to radiation exposure and by the matrix in which the cells were loaded with DCFH-DA substrate. Under the conditions optimized in this study, the DCF assay is capable of detecting increased DCFH oxidation in cell cultures irradiated with γ rays at a dose as low as 1.5 cGy. The increase in fluorescence was directly proportional to the radiation dose, which ranged from 0 to 2 Gy, and a minimal level of fluorescence was observed in sham-irradiated cells. These results indicate that the DCF assay optimized in this study is highly sensitive, linear and specific for measuring oxidative stress in irradiated cells.

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.

Development of the Bowman-Birk inhibitor for oral cancer chemoprevention and analysis of Neu immunohistochemical staining intensity with Bowman-Birk inhibitor concentrate treatment.

Objectives/Hypothesis: Cancer chemoprevention is a rapidly evolving approach to reverse or inhibit carcinogenesis, and there is active interest in development of effective chemopreventive agents against head and neck cancers. The retinoids are archetypal chemopreventive agents for oral premalignant lesions. They have significant clinical effect, but widespread use is limited by significant clinical toxicity. The Bowman‐Birk Inhibitor is one of several nontoxic compounds exhibiting both potent anticarcinogenic activity and minimal toxicity. The purposes of the study were to summarize the preclinical and clinical development of Bowman‐Birk Inhibitor and a Bowman‐Birk Inhibitor concentrate against oral premalignant lesions and to evaluate Neu immunohistochemical staining intensity for lesions and simultaneously obtained biopsy specimens of normal‐appearing mucosa from the Phase IIa Bowman‐Birk Inhibitor concentrate oral leukoplakia chemoprevention trial. Study Design: Part I is a selected literature review. Part II is a retrospective analysis of pathological specimens prospectively obtained from the Phase IIa clinical trial of Bowman‐Birk Inhibitor concentrate. Methods: Thirty‐two sets of biopsy specimens from lesions and uninvolved oral mucosa before and after treatment with Bowman‐Birk Inhibitor concentrate in doses ranging from 200 to 1066 chymotrypsin inhibitory units were examined in blinded fashion for Neu immunohistochemical staining intensity using the 3B‐5 monoclonal antibody. Staining intensity scores among the lesion and control biopsy specimens before and after Bowman‐Birk Inhibitor concentrate treatment were analyzed and compared with previously obtained values for serum Neu, oral mucosal cell Neu, protease activity, and clinical response to treatment. Results: Mean Neu staining score was significantly higher in lesions compared with uninvolved mucosa (P <.001). Pretreatment staining scores for biopsy specimens of lesions and control biopsy specimens of normal‐appearing tissues were correlated (Spearman correlation coefficient [r] = 0.375, P = .045), but no correlation between lesion and control biopsy specimen scores was evident after treatment. The change in Neu staining score with Bowman‐Birk Inhibitor concentrate treatment in control site biopsy specimens demonstrated an inverse relationship of change in lesion area with Bowman‐Birk Inhibitor concentrate treatment (Spearman r = −0.493, P <.007). Conclusion: Bowman‐Birk Inhibitor concentrate shows promise to become an effective nontoxic chemopreventive agent based on results of extensive preclinical studies, and Phase I and Phase IIa clinical trials. Bowman‐Birk Inhibitor concentrate has dose‐related clinical activity against oral leukoplakia and modulates levels of Neu and protease activity. The current investigation identified increased Neu staining intensity in hyperplastic lesions compared with simultaneously obtained biopsy specimens of normal‐appearing mucosa both before and after Bowman‐Birk Inhibitor concentrate treatment. This finding supports prior observations that increased Neu expression is present in a subset of oral premalignant lesions and head and neck cancers. The trend of increased Neu staining score in control biopsy tissues of subjects exhibiting decreased lesion area following Bowman‐Birk Inhibitor concentrate treatment raises questions about the mechanisms of Bowman‐Birk Inhibitor concentrate action. One possible explanation is that Bowman‐Birk Inhibitor stabilizes the extracellular domain of Neu, thereby preventing receptor truncation and internalization. Further study of modulation of Neu and protease activity by Bowman‐Birk Inhibitor concentrate treatment may provide insights into the role of proteases and protease inhibitors in oral premalignant lesions and the mechanisms underlying Bowman‐Birk Inhibitor concentrate effects. A Phase IIb randomized, placebo‐controlled clinical trial to determine the clinical effectiveness of Bowman‐Birk Inhibitor concentrate and further evaluate these candidate biomarkers is under way.

Effects of Bowman-Birk inhibitor on rat colon carcinogenesis.

The present study was undertaken to determine whether the Bowman-Birk inhibitor (BBI) could prevent colon carcinogenesis in rats treated with dimethylhydrazine (DMH) and whether there were adverse side effects associated with treatment with BBI for cancer prevention. BBI was evaluated in the forms of purified BBI (PBBI) or an extract of soybeans enriched in BBI, termed BBI concentrate (BBIC). The results demonstrate that PBBI and BBIC reduced the incidence and frequency of tumors in DMH-treated rats compared with animals treated with DMH alone. Autoclaved BBIC, in which the protease inhibitor activity of BBI was destroyed, had a weak and statistically insignificant, suppressive effect on DMH-induced colon carcinogenesis in rats, suggesting that the protease inhibitor activity of BBI is likely to be responsible for the anticarcinogenic activity of BBIC. Soy molasses, which contains soy isoflavones, did not have an effect on colon cancer carcinogenesis in DMH-treated rats. Similar to results from previous studies (Nauss et al. JNCI 73, 915-924, 1984), the most aggressive, malignant colon adenocarcinomas developed within or in association with gut-associated lymphoid tissue aggregates. No adverse side effects on the pancreas or animal growth were observed in rats treated with PBBI or BBIC. These results demonstrate that PBBI and BBIC may be used to prevent colon cancer without significant adverse side effects.

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.

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.

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.

Analysis of White Blood Cell Counts in Mice after Gamma- or Proton-Radiation Exposure

In the coming decades human space exploration is expected to move beyond low-Earth orbit. This transition involves increasing mission time and therefore an increased risk of radiation exposure from solar particle event (SPE) radiation. Acute radiation effects after exposure to SPE radiation are of prime importance due to potential mission-threatening consequences. The major objective of this study was to characterize the dose–response relationship for proton and &ggr; radiation delivered at doses up to 2 Gy at high (0.5 Gy/min) and low (0.5 Gy/h) dose rates using white blood cell (WBC) counts as a biological end point. The results demonstrate a dose-dependent decrease in WBC counts in mice exposed to high- and low-dose-rate proton and &ggr; radiation, suggesting that astronauts exposed to SPE-like radiation may experience a significant decrease in circulating leukocytes.

Detection of Oxidative Stress Induced by Low- and High-Linear Energy Transfer Radiation in Cultured Human Epithelial Cells

Abstract Wan, X. S., Bloch, P., Ware, J. H., Zhou, Z., Donahue, J. J., Guan, J., Stewart, J. and Kennedy, A. R. Detection of Oxidative Stress Induced by Low- and High-Linear Energy Transfer Radiation in Cultured Human Epithelial Cells. Radiat. Res. 163, 364–368 (2005). A standardized dichlorofluorescin (DCF) fluorometric assay capable of measuring radiation-induced oxidative stress was used to determine the effectiveness of protons and high-mass, high-atomic number (Z) and high-energy (HZE) particles to produce oxidative stress in vitro. Protons were found to be about equally as effective as X rays in the generation of oxidative stress in cultured cells. However, 56Fe-ion beams with energies of 1 GeV/nucleon and 5 GeV/nucleon were less effective than X rays or γ rays in inducing dichlorofluorescin (DCFH) oxidation. The relatively lower slope values for the dose responses of HZE-particle radiation-induced DCFH oxidation indicate that the sensitivity of the DCF fluorometric assay is probably dependent on the linear energy transfer (LET) of the radiation beam.

Dietary supplements reduce the cataractogenic potential of proton and HZE-particle radiation in mice.

Abstract The present study was undertaken to investigate the ability of dietary supplements to reduce the formation and severity of cataracts in mice irradiated with high-energy protons or iron ions, which are important components of the radiation encountered by astronauts during space travel. The mice were exposed to proton or iron-ion radiation and fed with a control diet or diets supplemented with the soybean-derived protease inhibitor, Bowman-Birk inhibitor (BBI), in the form of BBI Concentrate (BBIC) or an antioxidant formulation [containing l-selenomethionine (SeM), N-acetyl cysteine (NAC), ascorbic acid, co-enzyme Q10, α-lipoic acid and vitamin E succinate] both before and after the radiation exposure. At approximately 2 years after the radiation exposure, the animals were killed humanely and lenses were harvested and characterized using an established classification system that assigns discrete scores based on the severity of the lens opacifications. The results showed that exposure to 1 GeV/nucleon proton (3 Gy) or iron-ion (50 cGy) radiation significantly increased the cataract prevalence and severity in CBA/J mice to levels above the baseline levels of age-induced cataract formation in this mouse strain. Treatment with BBIC or the antioxidant formulation significantly reduced the prevalence and severity of the lens opacifications in the mice exposed to iron-ion radiation. Treatment with BBIC or the antioxidant formulation also decreased the severity of the lens opacifications in the mice exposed to proton radiation; however, the decrease did not reach statistical significance. These results indicate that BBIC and the antioxidant formulation evaluated in this study could be useful for protecting astronauts against space radiation-induced cataracts during or after long-term manned space missions.