Comparative analysis of colorimetric staining in skin using open-source software.

Colorimetric staining techniques such as immunohistochemistry (IHC), immunofluorescence (IF) and histochemistry (HC) provide useful information regarding the localization and relative amount of a molecule/substance in skin. We have developed a novel, straightforward method to assess colorimetric staining by combining features from two open-source software programs. As a proof of principle, we demonstrate the utility of this approach by analysing changes in skin melanin deposition during the radiation-induced tanning response of Yucatan mini-pigs. This method includes a visualization step to validate the accuracy of colour selection before quantitation to ensure accuracy. The data show that this method is robust and will provide a means to obtain accurate comparative analyses of staining in IHC/IF/HC samples.

Mitigating the risk of radiation-induced cancers: limitations and paradigms in drug development.

The United States radiation medical countermeasures (MCM) programme for radiological and nuclear incidents has been focusing on developing mitigators for the acute radiation syndrome (ARS) and delayed effects of acute radiation exposure (DEARE), and biodosimetry technologies to provide radiation dose assessments for guiding treatment. Because a nuclear accident or terrorist incident could potentially expose a large number of people to low to moderate doses of ionising radiation, and thus increase their excess lifetime cancer risk, there is an interest in developing mitigators for this purpose. This article discusses the current status, issues, and challenges regarding development of mitigators against radiation-induced cancers. The challenges of developing mitigators for ARS include: the long latency between exposure and cancer manifestation, limitations of animal models, potential side effects of the mitigator itself, potential need for long-term use, the complexity of human trials to demonstrate effectiveness, and statistical power constraints for measuring health risks (and reduction of health risks after mitigation) following relatively low radiation doses (<0.75 Gy). Nevertheless, progress in the understanding of the molecular mechanisms resulting in radiation injury, along with parallel progress in dose assessment technologies, make this an opportune, if not critical, time to invest in research strategies that result in the development of agents to lower the risk of radiation-induced cancers for populations that survive a significant radiation exposure incident.

Proteases, protease inhibitors and radiation carcinogenesis.

PURPOSE: The purpose of the studies described in this mini review article was to identify nontoxic compounds that could prevent or suppress the radiation induced malignant transformation of cells and be useful as human cancer preventive agents. CONCLUSIONS: (1) Many different types of potential anticarcinogenic substances were evaluated initially for their abilities to prevent or suppress radiation induced malignant transformation in vitro, and certain anticarcinogenic protease inhibitors (APIs) were observed to be the most powerful anticarcinogenic agents at suppressing this surrogate endpoint biomarker of radiation carcinogenesis. (2) Within the category of APIs, those that inhibited the activity of chymotrypsin were effective at far lower molar concentrations than other APIs. The soybean-derived protease inhibitor known as the Bowman-Birk inhibitor (BBI) is a particularly powerful chymotrypsin inhibitor that is able to prevent radiation induced transformation in vitro (at concentrations down to nanomolar levels) as well as radiation induced carcinogenesis in vivo without toxicity. (3) There were many other unusual characteristics of APIs that led to the selection of one of these APIs, BBI, as the most appropriate compound for us to develop as a human cancer preventive agent. As one example, the APIs have an irreversible effect on carcinogenesis, while the effects are reversible for most anticarcinogenic agents when they are removed from carcinogenesis assay systems. (4) Numerous studies were performed in attempts to determine the potential mechanisms by which the APIs could prevent or suppress radiation induced carcinogenesis in in vitro and in vivo systems, and the results of these studies are described in this review article. The APIs and the proteases which interact with them appear to play important roles in radiation carcinogenesis. (5) Preparations for human trials using BBI began decades ago. The cost of preparing purified BBI was far too high to consider performing human trials with this agent, so BBI Concentrate (BBIC), a soybean extract enriched in BBI, was developed for the specific purpose of performing human trials with BBI. BBIC achieved Investigational New Drug (IND) Status with the Food and Drug Administration in April,1992, and human BBIC trials began at that time. (6) Several human trials were performed using BBIC and they indicated many potentially beneficial health effects produced by BBIC administration to people in various forms (e.g. tablets). 7) It is hypothesized that BBI takes the place of α-1-antichymotrypsin, an important regulatory compound in the human body, and helps to maintain homeostasis.

Comparison of hindlimb unloading and partial weight suspension models for spaceflight-type condition induced effects on white blood cells.

Animal models are frequently used to assist in the determination of the long- and short-term effects of space flight. The space environment, including microgravity, can impact many physiological and immunological system parameters. It has been found that ground based models of microgravity produce changes in white blood cell counts, which negatively affects immunologic function. As part of the Center of Acute Radiation Research (CARR), we compared the acute effects on white blood cell parameters induced by the more traditionally used animal model of hindlimb unloading (HU) with a recently developed reduced weightbearing analog known as partial weight suspension (PWS). Female ICR mice were either hindlimb unloaded or placed in the PWS system at 16% quadrupedal weightbearing for 4 h, 1, 2, 7 or 10 days, at which point complete blood counts were obtained. Control animals (jacketed and non-jacketed) were exposed to identical conditions without reduced weightbearing. Results indicate that significant changes in total white blood cell (WBC), neutrophil, lymphocyte, monocyte and eosinophil counts were observed within the first 2 days of exposure to each system. These differences in blood cell counts normalized by day 7 in both systems. The results of these studies indicate that there are some statistically significant changes observed in the blood cell counts for animals exposed to both the PWS and HU simulated microgravity systems.

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.

Can radiation risks to patients be reduced without reducing radiation exposure? The status of chemical radioprotectants.

OBJECTIVE: Medical radiation exposure has increased sixfold since 1980 and is the largest controllable source of exposure. Many efforts have been devoted to reducing dose or eliminating unnecessary examinations but with limited success. The concern regarding nuclear terrorism has focused a large amount of attention on radioprotective drugs. The purpose of this article is twofold: to review the current concepts, potential, and limitations of chemical radioprotectants in reducing stochastic and deterministic effects and to assess the potential application to diagnostic and interventional medical radiation procedures. CONCLUSION: There are a wide variety of chemical compounds that have been studied for radioprotective effects. Although there is promising research, chemical radioprotectants have not been shown to be very effective and, with one limited exception, are not the standard of care in medicine.

Using electron beam radiation to simulate the dose distribution for whole body solar particle event proton exposure.

As a part of the near solar system exploration program, astronauts may receive significant total body proton radiation exposures during a solar particle event (SPE). In the Center for Acute Radiation Research (CARR), symptoms of the acute radiation sickness syndrome induced by conventional radiation are being compared to those induced by SPE-like proton radiation, to determine the relative biological effectiveness (RBE) of SPE protons. In an SPE, the astronaut's whole body will be exposed to radiation consisting mainly of protons with energies below 50 MeV. In addition to providing for a potentially higher RBE than conventional radiation, the energy distribution for an SPE will produce a relatively inhomogeneous total body dose distribution, with a significantly higher dose delivered to the skin and subcutaneous tissues than to the internal organs. These factors make it difficult to use a (60)Co standard for RBE comparisons in our experiments. Here, the novel concept of using megavoltage electron beam radiation to more accurately reproduce both the total dose and the dose distribution of SPE protons and make meaningful RBE comparisons between protons and conventional radiation is described. In these studies, Monte Carlo simulation was used to determine the dose distribution of electron beam radiation in small mammals such as mice and ferrets as well as large mammals such as pigs. These studies will help to better define the topography of the time-dose-fractionation versus biological response landscape for astronaut exposure to an SPE.

Plasma D-Dimer Levels are Elevated in Radiation Oncology Patients.

D-dimer plasma levels were evaluated to determine whether they are altered by radiation. D-dimer levels were measured in radiation oncology patients, who were diagnosed with prostate, breast or lung cancer, or leukemia, as well as in healthy subjects serving as controls. Blood samples from radiotherapy patients were taken at three different time points: pre-, on- and post-radiotherapy. For the patients, considered together, differences between the D-dimer levels at these three time points compared to controls were statistically significant. Compared to the pre-radiotherapy measurements, radiation exposure was associated with a significant increase in the D-dimer levels at the on- and post-radiotherapy time points. At the post-radiotherapy time point, D-dimer levels in the patients were not significantly reduced compared to the on-radiotherapy levels, indicating that the risk for developing disseminated intravascular coagulation (DIC) may be increased in some radiation oncology patients. Of particular concern are the post-radiotherapy results observed for the D-dimer levels in the leukemia patients, in which the average fold increase in the D-dimer levels was 5.43 (compared to the pre-radiotherapy levels). These results suggest that leukemia patients might benefit from frequent assessment of their D-dimer levels after their total-body irradiation-conditioning regimen to detect early signs of DIC development. It is hoped that the results described here will lead to heightened awareness in the radiation oncology community that the risk of DIC development is greatly increased in some of these patients.

Design, Implementation, and in Vivo Validation of a Novel Proton FLASH Radiation Therapy System.

PURPOSE: Recent studies suggest that ultrahigh-dose-rate, "FLASH," electron radiation therapy (RT) decreases normal tissue damage while maintaining tumor response compared with conventional dose rate RT. Here, we describe a novel RT apparatus that delivers FLASH proton RT (PRT) using double scattered protons with computed tomography guidance and provide the first report of proton FLASH RT-mediated normal tissue radioprotection. METHODS AND MATERIALS: Absolute dose was measured at multiple depths in solid water and validated against an absolute integral charge measurement using a Faraday cup. Real-time dose rate was obtained using a NaI detector to measure prompt gamma rays. The effect of FLASH versus standard dose rate PRT on tumors and normal tissues was measured using pancreatic flank tumors (MH641905) derived from the KPC autochthonous PanCa model in syngeneic C57BL/6J mice with analysis of fibrosis and stem cell repopulation in small intestine after abdominal irradiation. RESULTS: The double scattering and collimation apparatus was dosimetrically validated with dose rates of 78 ± 9 Gy per second and 0.9 ± 0.08 Gy per second for the FLASH and standard PRT. Whole abdominal FLASH PRT at 15 Gy significantly reduced the loss of proliferating cells in intestinal crypts compared with standard PRT. Studies with local intestinal irradiation at 18 Gy revealed a reduction to near baseline levels of intestinal fibrosis for FLASH-PRT compared with standard PRT. Despite this difference, FLASH-PRT did not demonstrate tumor radioprotection in MH641905 pancreatic cancer flank tumors after 12 or 18 Gy irradiation. CONCLUSIONS: We have designed and dosimetrically validated a FLASH-PRT system with accurate control of beam flux on a millisecond time scale and online monitoring of the integral and dose delivery time structure. Using this system, we found that FLASH-PRT decreases acute cell loss and late fibrosis after whole-abdomen and focal intestinal RT, whereas tumor growth inhibition is preserved between the 2 modalities.

Induction of cytokine gene expression in human thyroid epithelial cells irradiated with HZE particles (iron ions).

Gene expression profiles were examined using cDNA microarray technology in human thyroid epithelial (Htori-3) cells exposed to a low, non-toxic dose (10 cGy) of radiation from HZE particles in the form of iron ions in the absence or presence of selenomethionine (SeM). A total of 215 genes were differentially regulated 2 h after exposure to a 10-cGy dose of iron-ion radiation. In the microarray analysis, SeM had profound effects on the radiation-induced expression of several specific genes, which includes PLAU, IGFBP3, FOLR1, B4GALT1 and COL1A1. Of particular interest to us was a gene cluster, "secreted proteins", that was up-regulated after radiation exposure. Seven up-regulated genes of this gene cluster fall within the chemokine/cytokine gene cluster, namely, CXCL1, CXCL2, IL6, IL11, IL8, IL24 and TGFbeta2. In microarray studies, the radiation-induced up-regulated expression of some these genes encoding cytokine/chemokine proteins was significantly decreased by SeM treatment. For IL8, TGFbeta2, CXCL1 and CXCL2, these observations were validated by qPCR techniques. It is concluded that SeM can regulate ionizing radiation-induced gene expression and may serve as an effective countermeasure for some of the acute inflammatory/immune responses induced by low-dose HZE-particle radiation.

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

Abstract Dietary antioxidants have radioprotective effects after gamma-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, alpha-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 LD(50/30); 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 TGFbeta1 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.

Distinct vascular genomic response of proton and gamma radiation-A pilot investigation.

The cardiovascular biology of proton radiotherapy is not well understood. We aimed to compare the genomic dose-response to proton and gamma radiation of the mouse aorta to assess whether their vascular effects may diverge. We performed comparative RNA sequencing of the aorta following (4 hrs) total-body proton and gamma irradiation (0.5-200 cGy whole body dose, 10 dose levels) of conscious mice. A trend analysis identified genes that showed a dose response. While fewer genes were dose-responsive to proton than gamma radiation (29 vs. 194 genes; q-value ≤ 0.1), the magnitude of the effect was greater. Highly responsive genes were enriched for radiation response pathways (DNA damage, apoptosis, cellular stress and inflammation; p-value ≤ 0.01). Gamma, but not proton radiation induced additionally genes in vasculature specific pathways. Genes responsive to both radiation types showed almost perfectly superimposable dose-response relationships. Despite the activation of canonical radiation response pathways by both radiation types, we detected marked differences in the genomic response of the murine aorta. Models of cardiovascular risk based on photon radiation may not accurately predict the risk associated with proton radiation.

Effects of dietary antioxidant supplementation on the development of malignant lymphoma and other neoplastic lesions in mice exposed to proton or iron-ion radiation.

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.

Dietary antioxidants protect hematopoietic cells and improve animal survival after total-body irradiation.

The purpose of this study was to determine whether a dietary supplement consisting of L-selenomethionine, vitamin C, vitamin E succinate, alpha-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-beta1 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.

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 ViaLogy's (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.

Limitations in predicting the space radiation health risk for exploration astronauts.

Despite years of research, understanding of the space radiation environment and the risk it poses to long-duration astronauts remains limited. There is a disparity between research results and observed empirical effects seen in human astronaut crews, likely due to the numerous factors that limit terrestrial simulation of the complex space environment and extrapolation of human clinical consequences from varied animal models. Given the intended future of human spaceflight, with efforts now to rapidly expand capabilities for human missions to the moon and Mars, there is a pressing need to improve upon the understanding of the space radiation risk, predict likely clinical outcomes of interplanetary radiation exposure, and develop appropriate and effective mitigation strategies for future missions. To achieve this goal, the space radiation and aerospace community must recognize the historical limitations of radiation research and how such limitations could be addressed in future research endeavors. We have sought to highlight the numerous factors that limit understanding of the risk of space radiation for human crews and to identify ways in which these limitations could be addressed for improved understanding and appropriate risk posture regarding future human spaceflight.

Bowman-Birk inhibitor concentrate prevents atrophy, weakness, and oxidative stress in soleus muscle of hindlimb-unloaded mice.

Antigravity muscles atrophy and weaken during prolonged mechanical unloading caused by bed rest or spaceflight. Unloading also induces oxidative stress in muscle, a putative cause of weakness. We tested the hypothesis that dietary supplementation with Bowman-Birk inhibitor concentrate (BBIC), a soy protein extract, would oppose these changes. Adult mice were fed a diet supplemented with 1% BBIC during hindlimb unloading for up to 12 days. Soleus muscles of mice fed the BBIC-supplemented diet weighed less, developed less force per cross-sectional area, and developed less total force after unloading than controls. BBIC supplementation was protective, blunting decrements in soleus muscle weight and force. Cytosolic oxidant activity was assessed using 2',7'-dichlorofluorescin diacetate. Oxidant activity increased in unloaded muscle, peaking at 3 days and remaining elevated through 12 days of unloading. Increases in oxidant activity correlated directly with loss of muscle mass and were abolished by BBIC supplementation. In vitro assays established that BBIC directly buffers reactive oxygen species and also inhibits serine protease activity. We conclude that dietary supplementation with BBIC protects skeletal muscle during prolonged unloading, promoting redox homeostasis in muscle fibers and blunting atrophy-induced weakness.

Establishment of human cancer cell clones with different characteristics: a model for screening chemopreventive agents.

BACKGROUND: The present study was undertaken in order to establish phenotypically different cell clones from 10 parental lines of human breast (MCF-7 and T-47D), prostate (PC-3 and DU145), lung (A549 and A427), colon (HCT-116 and HT-29) and bladder (TCCSUP and T24) cancer cells. MATERIALS AND METHODS: Sublines were established from each of the parental lines by the limiting dilution method. The derived clones were characterized in terms of plating efficiency, cell proliferation rate, saturation density and colony formation efficiency in soft agar. RESULTS: Phenotypically different cell clones were derived from each parental human cancer cell line, with many clones having more 'normal' characteristics than the parental line from which they were derived. CONCLUSION: Phenotypically normal clones obtained through clonal selection from human cancer cell populations are expected to be a useful tool for the screening of cancer chemopreventive agents and the study of tumor progression.

Protection against radiation-induced oxidative stress in cultured human epithelial cells by treatment with antioxidant agents.

PURPOSE: To evaluate the protective effects of antioxidant agents against space radiation-induced oxidative stress in cultured human epithelial cells. METHODS AND MATERIALS: The effects of selected concentrations of N-acetylcysteine, ascorbic acid, sodium ascorbate, co-enzyme Q10, alpha-lipoic acid, l-selenomethionine, and vitamin E succinate on radiation-induced oxidative stress were evaluated in MCF10 human breast epithelial cells exposed to radiation with X-rays, gamma-rays, protons, or high mass, high atomic number, and high energy particles using a dichlorofluorescein assay. RESULTS: The results demonstrated that these antioxidants are effective in protecting against radiation-induced oxidative stress and complete or nearly complete protection was achieved by treating the cells with a combination of these agents before and during the radiation exposure. CONCLUSION: The combination of antioxidants evaluated in this study is likely be a promising countermeasure for protection against space radiation-induced adverse biologic effects.