Zhaozong Zhou

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.

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.

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.

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.

Protection against Adverse Biological Effects Induced by Space Radiation by the Bowman-Birk Inhibitor and Antioxidants

Abstract Kennedy, A. R., Zhou, Z., Donahue, J. J. and Ware, J. H. Protection against Adverse Biological Effects Induced by Space Radiation by the Bowman-Birk Inhibitor and Antioxidants. Radiat. Res. 166, 327–332 (2006). This study was undertaken to evaluate the protective effects of the soybean-derived Bowman-Birk inhibitor (BBI), BBI concentrate (BBIC) and/or antioxidants against the adverse biological effects induced by space radiation in cultured human epithelial cells. The effects of BBI, BBIC and a combination of ascorbic acid, co-enzyme Q10, l-selenomethionine (SeM) and vitamin E succinate on proton and HZE-particle [high-energy (high E) nuclei of heavier (high atomic number, Z) elements] radiation-induced cytotoxicity in MCF10 human breast epithelial cells and a phenotypic change associated with transformation in HTori-3 human thyroid epithelial cells were assessed with a clonogenic survival assay and a soft agar colony formation assay. The results demonstrate that BBIC and antioxidants are effective in protecting against space radiation-induced cytotoxicity in MCF10 cells and BBI, BBIC and antioxidants are effective in protecting against a space radiation-induced phenotypic change associated with transformation of HTori-3 cells.

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.

Effects of Selenomethionine in Irradiated Human Thyroid Epithelial Cells and Tumorigenicity Studies

The objectives of the present study were to characterize γ-ray, 1 GeV/n proton, and 1 GeV/n iron ion radiation-induced adverse biological effects in terms of toxicity and transformation of HTori-3 human thyroid epithelial cells; to evaluate the ability of L-selenomethionine (SeM) to protect against radiation-induced transformation when present at different times during the assay period; and to evaluate the tumorigenicity of HTori-3 cells derived from anchorage-independent colonies following iron ion radiation exposure. Cell survival was determined by a clonogenic assay, transformation was measured by a soft agar colony formation assay, and the tumorigenic potential of the cells was determined by injecting them subcutaneously into athymic nude mice and monitoring tumor formation. The results demonstrate that exposure of HTori-3 cells to γ-ray, proton, or iron ion radiation resulted in decreased clonogenic survival, which persisted for weeks after the radiation exposure. Treatment with SeM initiated up to 7 days after the radiation exposure conferred significant protection against radiation-induced anchorage-independent growth. HTori-3 cells derived from all evaluated anchorage-independent colonies formed tumors when injected into athymic nude mice, indicating that these cells are tumorigenic and that anchorage-independent colony growth is a reliable surrogate endpoint biomarker for the radiation-induced malignant transformation of HTori-3 cells.

Effects of selenomethionine on the gene expression profile of cloned human prostate cancer cells representing a phenotypic continuum of cancer progression.

We previously characterized three cell clones that were derived by limiting dilution from a human prostate cancer cell line (LNCaP) representing a phenotypic continuum of cancer progression (1). The present study was undertaken to examine the effects of L-selenomethionine (SeM), a potential cancer chemopreventive agent, on the gene expression profile of the cultured cell clones. Following a three-day incubation period with SeM, total RNA was extracted, and the gene expression profile was evaluated using Affymetrix human HG U133A microarrays and analyzed by ViaLogys (Altadena, CA) VMAxS® platform deploying quantum resonance interferometry (QRI) processing. The differentially expressed genes and corresponding biological processes were compared across the different treatments and cell types. Whereas SeM significantly affected RNA–DNA metabolism and protein transport and metabolism in all of the cell types evaluated, significant effects of SeM on genes mainly involved in the pathways of cell cycle, growth, differentiation, and apoptosis were observed only in the cell clone with a more malignant phenotype.