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.

5-AED enhances survival of irradiated mice in a G-CSF-dependent manner, stimulates innate immune cell function, reduces radiation-induced DNA damage and induces genes that modulate cell cycle progression and apoptosis.

The steroid androst-5-ene-3ß,17ß-diol (5-androstenediol, 5-AED) elevates circulating granulocytes and platelets in animals and humans, and enhances survival during the acute radiation syndrome (ARS) in mice and non-human primates. 5-AED promotes survival of irradiated human hematopoietic progenitors in vitro through induction of Nuclear Factor-κB (NFκB)-dependent Granulocyte Colony-Stimulating Factor (G-CSF) expression, and causes elevations of circulating G-CSF and interleukin-6 (IL-6). However, the in vivo cellular and molecular effects of 5-AED are not well understood. The aim of this study was to investigate the mechanisms of action of 5-AED administered subcutaneously (s.c.) to mice 24 h before total body γ- or X-irradiation (TBI). We used neutralizing antibodies, flow cytometric functional assays of circulating innate immune cells, analysis of expression of genes related to cell cycle progression, DNA repair and apoptosis, and assessment of DNA strand breaks with halo-comet assays. Neutralization experiments indicated endogenous G-CSF but not IL-6 was involved in survival enhancement by 5-AED. In keeping with known effects of G-CSF on the innate immune system, s.c. 5-AED stimulated phagocytosis in circulating granulocytes and oxidative burst in monocytes. 5-AED induced expression of both bax and bcl-2 in irradiated animals. Cdkn1a and ddb1, but not gadd45a expression, were upregulated by 5-AED in irradiated mice. S.c. 5-AED administration caused decreased DNA strand breaks in splenocytes from irradiated mice. Our results suggest 5-AED survival enhancement is G-CSF-dependent, and that it stimulates innate immune cell function and reduces radiation-induced DNA damage via induction of genes that modulate cell cycle progression and apoptosis.

The U.S. government's medical countermeasure portfolio management for nuclear and radiological emergencies: synergy from interagency cooperation.

Following the attacks of 11 September 2001, emergency preparedness within the U.S. Department of Health and Human Services, as well as at the Department of Defense and other federal agencies, received higher visibility, new mandates and increased funding. Emergency deployment teams increased the frequency of drills to enable better response to the health consequences of mass-casualty incidents. Interagency coordination has also continued to increase to more efficiently and effectively leverage federal resources toward emergency medical preparedness for both civilian and military populations.

Synopsis of partial-body radiation diagnostic biomarkers and medical management of radiation injury workshop.

Radiation exposures from accidents, nuclear detonations or terrorist incidents are unlikely to be homogeneous; however, current biodosimetric approaches are developed and validated primarily in whole-body irradiation models. A workshop was held at the Armed Forces Radiobiology Research Institute in May 2008 to draw attention to the need for partial-body biodosimetry, to discuss current knowledge, and to identify the gaps to be filled. A panel of international experts and the workshop attendees discussed the requirements and concepts for a path forward. This report addresses eight key areas identified by the Workshop Program Committee for future focus: (1) improved cytogenetics, (2) clinical signs and symptoms, (3) cutaneous bioindicators, (4) organ-specific biomarkers, (5) biophysical markers of dose, (6) integrated diagnostic approaches, (7) confounding factors, and (8) requirements for post-event medical follow-up. For each area, the status, advantages and limitations of existing approaches and suggestions for new directions are presented.

Rapid radiation dose assessment for radiological public health emergencies: roles of NIAID and BARDA.

A large-scale radiological incident would result in an immediate critical need to assess the radiation doses received by thousands of individuals to allow for prompt triage and appropriate medical treatment. Measuring absorbed doses of ionizing radiation will require a system architecture or a system of platforms that contains diverse, integrated diagnostic and dosimetric tools that are accurate and precise. For large-scale incidents, rapidity and ease of screening are essential. The National Institute of Allergy and Infectious Diseases of the National Institutes of Health is the focal point within the Department of Health and Human Services (HHS) for basic research and development of medical countermeasures for radiation injuries. The Biomedical Advanced Research and Development Authority within the HHS Office of the Assistant Secretary for Preparedness and Response coordinates and administers programs for the advanced development and acquisition of emergency medical countermeasures for the Strategic National Stockpile. Using a combination of funding mechanisms, including funds authorized by the Project BioShield Act of 2004 and those authorized by the Pandemic and All-Hazards Preparedness Act of 2006, HHS is enhancing the nation's preparedness by supporting the radiation dose assessment capabilities that will ensure effective and appropriate use of medical countermeasures in the aftermath of a radiological or nuclear incident.

Effects of genistein administration on cytokine induction in whole-body gamma irradiated mice.

The development of an effective pharmacological countermeasure is needed to reduce the morbidity and mortality in military and civilian populations associated with possible exposure to ionizing radiation. We previously demonstrated that a single subcutaneous (sc) administration of genistein at a non-toxic dose provided protection against acute radiation injury and that the radioprotective effects were associated with multilineage, hematopoietic progenitor cell recovery. The purpose of this study was to determine whether hematopoietic recovery was preceded by cytokine induction. In mice treated with sc genistein 24 h before irradiation (7 Gy 60Co), we quantified serum cytokine levels by multiplex Luminex and also investigated a larger number of cytokines using cytokine arrays. Genistein administration stimulated serum granulocyte-colony stimulating factor (G-CSF) 4h and 24h after sham irradiation or gamma-irradiation. Interleukin-6 (IL-6) was significantly increased in genistein-treated animals 4h after irradiation. Because G-CSF and IL-6 are important hematopoietic factors, these results support our hypothesis that the previously observed radioprotective efficacy by genistein may be a result of early recovery of hematopoietic cells due to enhanced production of G-CSF and IL-6.

Use of a centrifuge-based automated blood cell counter for radiation dose assessment.

Hematological changes create early-response biomarkers for assessing radiation doses. Existing dose-prediction models are based on serial blood lymphocyte counts after acute whole-body exposure to gamma-radiation. Measurements of lymphocyte-depletion kinetics after possible exposures are useful for triaging patients and managing medical resources. The small-footprint QBC Autoread Plus System provides cost-effective hematological analyses with reproducibility, accuracy, and a broad dynamic range. QBC analysis measures centrifugally packed, whole blood cells in microhematocrit tubes and reports pooled lymphocyte and monocyte counts. Our objective was to modify this procedure to report pure lymphocyte counts for radiation biodosimetry applications. The CD14 antigen is strongly expressed on most human monocytes. Using anti-CD14-coated Dynabeads, we have devised a rapid method for depleting monocytes from whole blood without altering the lymphocyte viability or count. This simple dry procedure provides reliable lymphocyte counts for results that fall within the normal lymphocyte count range (1-4 x 10(9) cells per L) for radiation exposure assessment using lymphocyte-depletion kinetics.

Human in vivo radiation-induced biomarkers: gene expression changes in radiotherapy patients.

After initially identifying potential biomarkers of radiation exposure through microarray studies of ex vivo irradiated human peripheral white blood cells, we have now measured the in vivo responses of several of these biomarker genes in patients undergoing total body irradiation. Microarray analysis has identified additional in vivo radiation-responsive genes, although the general in vivo patterns of stress-gene induction appear similar to those obtained from ex vivo white blood cell experiments. Additional studies may reveal correlations between responses and either diagnosis or prognosis, and such in vivo validation marks an important step in the development of potentially informative radiation exposure biomarkers.

Development and assessment of a quantitative reverse transcription-PCR assay for simultaneous measurement of four amplicons.

BACKGROUND: High-throughput and forward-deployable biological dosimetry capabilities are required for tactical and medical decisions after radiologic events. We previously reported a quantitative reverse transcription (QRT)-PCR assay for human radiation-responsive gene targets using a whole-blood ex vivo irradiation model, but we needed a multitarget assay on a smaller, less costly, real-time PCR detection system. METHODS: We developed a quadruplex QRT-PCR assay in a 96-well, closed-plate format suitable for use with RNA extracted from whole blood. Four cDNA targets were simultaneously amplified in a sealed tube by hybridization to exonuclease probes, each conjugated to distinct fluorogenic reporters. A novel primer-limited 18S rRNA reference target was validated from serial dilutions of human total RNA. To test assay precision, we incorporated a positive-control cDNA mimic into duplex and quadruplex PCR reactions. The master mixture was supplemented with more enzyme, MgCl(2), and deoxyribonucleotides. Simultaneous detection of four targets was evaluated in comparison with respective duplex QRT-PCR assays. RESULTS: The simultaneous detection of three radiation-responsive genes by quadruplex QRT-PCR was quantitative, with gene expression changes similar to those observed with optimized duplex and triplex QRT-PCR assays. The 18S rRNA and GADD45 calibration curves (threshold cycle vs log(10) cDNA) were linear and reproducible and showed optimal PCR efficiencies as indicated by slopes statistically equivalent to the theoretical value of -3.322. CONCLUSIONS: This is the first study of a quadruplex QRT-PCR assay. Our approach has diagnostic utility in the detection of biomarkers, biological and toxicologic agents, and genes of inherited diseases and cancer.