[en] Chromosome aberration analysis is the conventional means of assessing radiation exposure. The Armed Forces Radiobiological Research Institute recently established an alternative method to measure radiation-induced chromosome aberrations in interphase cells. The method uses commercially available chemical agents to induce premature chromosome condensation in 'resting' G₀ human peripheral blood lymphocytes. Then specific whole-chromosome DNA probes are used with fluorescence in situ hybridisation to detect aberrant cells rapidly over a broad dose range. In new research, the real-time fluorogenic 5'-nuclease, or TaqMan^TM, polymerase chain reaction assay is being used to identify radiation-responsive molecular biomarkers, including gene expression targets and DNA mutations. The goal is to establish rapid, precise, high-throughput assay systems that are practical in a variety of radiation exposure scenarios. The new methodologies that have a number of other applications, together with diagnostic software now in development, could improve the United States military's emergency response capability and medical readiness. (author)

[en] Effective medical management of a suspected radiation overexposure incident necessitates recording dynamic medical data and measuring appropriate radiation bioassays in order to provide diagnostic information to the treating physician and dose assessment for the personnel radiation protection records. Biological Assessment Tool (BAT), a radiation casualty management software application available at the AFRRI's website (www.afrri.usuhs.mil), was developed to facilitate medical recording and to assist in bioassay dose predictions. Prodromal signs and symptoms as well as the body location and degree of transitory radiation-induced erythema should be recorded. Goans and colleagues have recently published dose prediction models for the prodromal symptom, time until onset of vomiting, following photon and criticality accident exposures. A complete blood cell count (CBC) with white cell differential should be obtained immediately after exposure, three times a day for the next 2-3 days, and then twice a day for the following 3- 6 days. Lymphocyte cell counts and lymphocyte depletion kinetics provide a dose assessment prediction between 1 and 10 Gy photon equivalent dose range. A blood sample should also be taken for dose assessment, following coordination with a qualified radiation cytogenetic biodosimetry laboratory, typically 24 hours after exposure using the 'gold standard' lymphocyte-dicentric radiation bioassay. Several of these cytogenetic biodosimetry laboratories use the premature chromosome condensation (PCC) bioassay, which permits dose assessment at higher doses (>5 Gy photon equivalent and acute high-dose rate exposures). Other opportunistic dosimetry approaches should be considered if available. Automated instrumentation to accomplish sample preparation and analysis for the conventional cytogenetic-based bioassays need to be integrated and beta tested in reference cytogenetic biodosimetry laboratories. Complimentary novel interphase-based cytological bioassays that detect cells with chromosomal type aberrations and radiation responsive molecular biomarkers (i.e., gene expression, protein) need to be validated and optimized for rapid radiation dose assessment applications

[en] The alkaline/filter DNA elution technique measure single-strand DNA breaks in mammalian cells based on the DNA molecular weight dependent retention of the macromolecule on 2-μm-pore-size filters. Described here is a modification of the technique which uses [₃H]thymidine-labeled DNA of ß-irradiated cells as an internal reference. Thus, an increased precision is obtained in the assessment of this type of DNA damage at biologically significant radiation doses (i.e., where cell survival occurs). The measure of DNA damage is based on the actual initial DNA elution rate, i.e., arithmetic ratio of the elution of ''test'' DNA (i.e., ₁₄C-labeled DNA) relative to the elution of ''reference'' DNA (i.e., ₃H-labeled DNA). The repair of this damage on postirradiation incubation of the cells is detected as a decrease in the rate of ''test'' DNA eluted relative to ''reference'' DNA from unincubated cells. For Chinese hamster V79-171 cells irradiated with 5 Gy (500 rads), repair can be resolved into two first-order processes having rate constants (at 24₀C) of approx.0.190 and approx.0.017 min^-₁

[en] Cell killing by ionizing radiation has been shown to be caused by hydroxyl free radicals formed by water radiolysis. The authors have previously suggested that the killing is not caused by individual OH free radicals but by the interaction of volumes of high radical density with DNA to cause locally multiple damaged sites (LMDS). Here they test this hypothesis using hydrogen dioxide as an alternate source of OH radicals. The route to OH production from H₂O₂ is expected to cause singly damaged sites rather than LMDS. Chinese hamster V79-171 cells were treated with H₂O₂ at varying concentrations for varying times at 0⁰C. The yield of DNA damage produced increases with increasing concentration of H₂O₂ and with time of exposure. H₂O₂ is efficient in producing single-strand breaks; treatment with 50 μM for 30 min produces damage equivalent to that formed by 10 Gy of α irradiation. In the presence of a hydroxyl radical scavenger, dimethyl sulfoxide (DMSO), the yield of damage decreases with increasing DMSO concentration consistent with the scavenging of hydroxyl radicals. In contrast to DNA damage production, cell killing by H₂O₂ treatment at 0⁰C is inefficient. The conclusion drawn is that individual DNA damage sites are ineffectual in killing cells. Mechanisms are suggested for killing at 0⁰C at high concentrations and for the efficient cell killing by H₂O₂ at 37⁰C at much lower concentrations

[en] Using a model system of in vitro human peripheral blood lymphocytes, the effect of low-dose (0.25 to 1.50 Gy) 250-kV_p X ray radiation (1 Gy.min^-1) on the expression of several proto-oncogenes was examined (c-Haras, c-src, c-met, c-jun, c-fos, and c-myc) and β-actin from 0.25 to 17 h post-radiation. RNA was extracted from cells harvested at various times after exposure and examined for levels of particular mRNAs by northern blot hybridisation. A progressive time- and dose-dependent increase in mRNA levels was observed for c-Haras mRNA, while the other proto-oncogenes (c-src, c-met, c-fos, c-jun, and c-myc) examined were variable during the same time period. β-actin levels were initially decreased but at 17 h post-radiation had returned to control levels. A comparison of the rate of c-Haras transcription at 5 and 17 h post-irradiation revealed that c-Haras transcription was higher at 5 h than at 17 h. These findings suggest that the level of specific proto-oncogene expression, particularly c-Haras, may be useful early diagnostic molecular biomarkers for biodosimetry applications. The use of real-time PCR technologies to quantify gene expression changes will also be discussed. (author)

[en] Short communication

[en] The exposure to hydrogen peroxide (H/sub 2/O/sub 2/) has been examined as a possible radiomimetic treatment for the study of radiation damage in cultured mammalian cells. The chemical nature of this challenge, most likely a Fenton-type reaction mediated by hydroxyl radicals, should result in a lower probability of multiply damaged sites when compared with ionizing radiation at equal levels of molecular events. Further, by carrying out this reaction at near ice-bath temperatures, the accumulation of H/sub 2/O/sub 2/-induced damage occurs while temperature-dependent ''repair'' processes should be inhibited in large part. In order to gain insight into mechanisms of radiation-induced inactivation of mammalian cells, the authors have extended previous H/sub 2/O/sub 2/-studies to include additional cellular endpoints. The loss of colony forming ability of Chinese hamster V79-AO3 cells following exposure to radiation, H/sub 2/O/sub 2/, and/or other drugs was measured. Exponentially growing cells were exposed to H/sub 2/O/sub 2/ at 4-9⁰C for 30 minutes. Dose response determinations for asynchronous cells treated in suspension or in monolayer culture condition have shown that killing occurs with a threshold of approximately 50 μM. At 50 mM H/sub 2/O/sub 2/ the surviving percentage was approximately 60%. Current studies are underway to examine the effect of higher concentrations of H/sub 2/O/sub 2/ on cell lethality. In addition, synchronous growing cells exposed to H/sub 2/O/sub 2/show some structure in the age response curve. Radical scavengers, which have been shown to spare H/sub 2/O/sub 2/-induced DNA damage, are being tested in this model system as to their effect in modifying H/sub 2/O/sub 2/-induced killing

[en] Prostaglandins (PG's) are highly active substances with a wide range of reported effects. One of these prostaglandins, PGE-2, has been demonstrated to be a radioprotective agent for some cell lines when irradiated in vitro. The authors examined the dependency of intracellular thiol content, modified by thiol binding or synthesis inhibitors, on the mechanism(s) of this cellular radioprotective effect. They treated exponentially growing synchronous and asynchronous Chinese hamster fibroblasts with both PGE-2 and N-ethyl-maleimide (NEM) to determine how PGE-2 affects cellular radiosensitivity. Chinese hamster V79-AO3 cells were incubated in alpha MEM medium supplemented with 10% fetal bovine serum. A two hr pretreatment with 14 μM PGE-2 in medium did not radioprotect the cells from graded doses of x-rays (Do = 2.0, Gy, Dq = 5.7 Gy). However, when the cells were exposed to a nonspecific thiol binding radiosensitizer, 1 μM NEM in phosphate buffered saline for 30 min, PGE-2 radioprotected these cells. This radioprotection was expressed as a 0.8 to 1.3 Gy increase in the shoulder of the asynchronous radiation survival curve. This increased survival, approximately 2-fold for asynchronous cells after 10 Gy, was also demonstrated in cells selected in mitosis and irradiated in G1 or S phase. Incubation in medium with 0.30 mM buthionine-S, R-sulfoximine (BSO) did not inhibit this recovery thus suggesting that this recovery might not be glutathione mediated

[en] It has generally been accepted that most biologically derived agents that are radioprotective in the hemopoietic-syndrome dose range (eg, endotoxin, Bacillus Calmette Guerin, Corynebacterium parvum, etc) exert their beneficial properties by enhancing hemopoietic recovery and hence, by regenerating the host's ability to resist life-threatening opportunistic infections. However, using glucan as a hemopoietic stimulant/radioprotectant, we have demonstrated that host resistance to opportunistic infection is enhanced in these mice even prior to the detection of significant hemopoietic regeneration. This early enhanced resistance to microbial invasion in glucan-treated irradiated mice could be correlated with enhanced and/or prolonged macrophage (but not granulocyte) function. These results suggest that early after irradiation glucan may mediate its radioprotection by enhancing resistance to microbial invasion via mechanisms not necessarily predicated on hemopoietic recovery. In addition, preliminary evidence suggests that glucan can also function as an effective free-radical scavenger. Because macrophages have been shown to selectively phagocytize and sequester glucan, the possibility that these specific cells may be protected by virtue of glucan's scavenging ability is also suggested

[en] Gas chromatography-mass spectrometry with selected-ion monitoring was used to study radiation-induced damage to DNA. Quantitative analysis of modified purine and pyrimidine bases resulting from exposure to ionizing radiation using this technique is dependent upon the selection of appropriate internal standards and calibration of the mass spectrometer for its response to known quantities of the internal standards and the products of interest. The compounds 6-azathymine and 8-azaadenine were found to be suitable internal standards for quantitative measurement of base damage in DNA. For the purpose of calibration of the mass spectrometer relative molar response factors for intense characteristic ions were determined for the trimethylsilyl derivatives of 5-hydroxyuracil, thymine glycol, and 5,6-dihydrothymine using 6-azathymine, and for the trimethylsilyl derivatives of 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine using 8-azaadenine. Accurate measurement of the yield of radiation-induced modifications to the DNA bases is also dependent upon two chemical steps in which the purines and pyrimidines are released from the sugar-phosphate backbone and then derivatized to make them volatile for gas chromatography. The completeness of these reactions, in addition to assessing the stability of the modified DNA bases in acid and their trimethylsilylated derivatives over the time necessary to complete the experimental analysis was also examined. Application of this methodology to the measurement of radiation-induced base modification in heat-denatured, nitrous oxidesaturated aqueous solutions of DNA is presented