The Internet's role in a biodosimetric response to a radiation mass casualty event.

Response to a large-scale radiological incident could require timely medical interventions to minimize radiation casualties. Proper medical care requires knowing the victim's radiation dose. When physical dosimetry is absent, radiation-specific chromosome aberration analysis can serve to estimate the absorbed dose in order to assist physicians in the medical management of radiation injuries. A mock exercise scenario was presented to six participating biodosimetry laboratories as one individual acutely exposed to Co under conditions suggesting whole-body exposure. The individual was not wearing a dosimeter and within 2-3 h of the incident began vomiting. The individual also had other medical symptoms indicating likelihood of a significant dose. Physicians managing the patient requested a dose estimate in order to develop a treatment plan. Participating laboratories in North and South America, Europe, and Asia were asked to evaluate more than 800 electronic images of metaphase cells from the patient to determine the dicentric yield and calculate a dose estimate with 95% confidence limits. All participants were blind to the physical dose until after submitting their estimates based on the dicentric chromosome assay (DCA). The exercise was successful since the mean biological dose estimate was 1.89 Gy whereas the actual physical dose was 2 Gy. This is well within the requirements for guidance of medical management. The exercise demonstrated that the most labor-intensive step in the entire process (visual evaluation of images) can be accelerated by taking advantage of world-wide expertise available on the Internet.

Analysis of chromosome damage for biodosimetry using imaging flow cytometry.

The dicentric chromosome assay (DCA), which involves counting the frequency of dicentric chromosomes in mitotic lymphocytes and converting it to a dose-estimation for ionizing radiation exposure, is considered to be the gold standard for radiation biodosimetry. Furthermore, for emergency response, the DCA has been adapted for triage by simplifying the scoring method [1]. With the development of new technologies such as the imaging flow cytometer, it may now be possible to adapt this microscope-based method to an automated cytometry method. This technology allows the sensitivity of microscopy to be maintained while adding the increased throughput of flow cytometry. A new protocol is being developed to adapt the DCA to the imaging cytometer in order to further increase the rapid determination of a biological dose. Peripheral blood mononuclear cells (PBMC) were isolated from ex vivo irradiated whole blood samples using a density gradient separation method and cultured with PHA and Colcemid. After 48h incubation, the chromosomes were isolated, stained for DNA content with propidium iodide (PI) and labelled with a centromere marker. Stained chromosomes were then analyzed on the ImageStream(×) (EMD-Millipore, Billerica, MA). Preliminary results indicate that individual chromosomes can be identified and mono- and dicentric chromosomes can be differentiated by imaging cytometry. A dose response curve was generated using this technology. The details of the method and the dose response curve are presented and compared to traditional microscope scoring. Imaging cytometry is a new technology which enables the rapid, automated analysis of fluorescently labelled chromosomes. Adapting the dicentric assay to this technology has the potential for high throughput analysis for mass casualty events.

Evaluating the biological effects of intermittent 1.9 GHz pulse-modulated radiofrequency fields in a series of human-derived cell lines.

Several recent studies have suggested that radiofrequency (RF) fields may cause changes in a variety of cellular functions that may eventually lead to potential long-term health effects. In the present study, we have assessed the ability of non-thermal RF-field exposure to affect a variety of biological processes (including apoptosis, cell cycle progression, viability and cytokine production) in a series of human-derived cell lines (TK6, HL60 and Mono-Mac-6). Exponentially growing cells were exposed to intermittent (5 min on, 10 min off) 1.9 GHz pulse-modulated RF fields for 6 h at mean specific absorption rates (SARs) of 0, 1 and 10 W/kg. Concurrent negative (incubator) and positive (heat shock for 1 h at 43 degrees C) controls were included in each experiment. Immediately after the 6-h exposure period and 18 h after exposure, cell pellets were collected and analyzed for cell viability, the incidence of apoptosis, and alterations in cell cycle kinetics. The cell culture supernatants were assessed for the presence of a series of human inflammatory cytokines (TNFA, IL1B, IL6, IL8, IL10, IL12) using a cytometric bead array assay. No detectable changes in cell viability, cell cycle kinetics, incidence of apoptosis, or cytokine expression were observed in any of RF-field-exposed groups in any of the cell lines tested, relative to the sham controls. However, the positive (heat-shock) control samples displayed a significant decrease in cell viability, increase in apoptosis, and alteration in cell cycle kinetics (G(2)/M block). Overall, we found no evidence that non-thermal RF-field exposure could elicit any detectable biological effect in three human-derived cell lines.

Broad-spectrum sunscreens prevent the secretion of proinflammatory cytokines in human keratinocytes exposed to ultraviolet A and phototoxic lomefloxacin.

The combination of phototoxic drugs and ultraviolet (UV) radiation can trigger the release of proinflammatory cytokines. The present study measured the ability of sunscreens to prevent cytokine secretion in human keratinocytes following cotreatment of these cells with a known photoreactive drug and UVA. Keratinocytes were treated for 1 h with increasing concentrations of lomefloxacin (LOM) or norfloxacin (NOR), exposed to 15 J/cm2 UVA, and incubated for 24 h. NOR, owing to the absence of a fluorine atom in position 8, was non-phototoxic and used as a negative control. Cell viability and the release of 3 cytokines were assessed, namely interleukin-1alpha (IL-1alpha), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNF-alpha). The measurement of these cytokines may be a useful tool for detecting photoreactive compounds. To measure their ability to prevent cytokine secretion, various sunscreens were inserted between the UVA source and the cells. Treatment with NOR, NOR plus UVA, or LOM had no effect on the cells. LOM plus UVA, however, had an effect on cell viability and on cytokine secretion. IL-1alpha levels increased with LOM concentration. The release of TNF-alpha and IL-6 followed the same pattern at lower concentrations of LOM but peaked at 15 micromol/L and decreased at higher concentrations. Sunscreens protected the cells from the effects of LOM plus UVA, as cell viability and levels of cytokines remained the same as in the control cells. In conclusion, the application of broad-spectrum sunscreen by individuals exposed to UVA radiation may prevent phototoxic reactions initiated by drugs such as LOM.

Exposure to chronic noise and fractionated X-ray radiation elicits biochemical changes and disrupts body weight gain in rat.

The aim was to assess the developmental and biochemical effects resulting from separate and combined exposures to radiation and noise in adult male Sprague-Dawley rats. For 21 days, animals were exposed daily (1) to whole-body 121 kVp X-ray exposure (cumulative dose=5 Gy), (2) to random intermittent noise band-limited between 0.4 and 20 kHz; 2 h day(-1) 86 decibels (dB) and (3) to combined exposures. Control animals were housed under ambient noise conditions 55 dB A-weighted (dBA) and sham-exposed to X-rays. Body weight gain was significantly reduced in animals exposed to either X-rays or noise, and the loss was more pronounced in animals exposed to both conditions. Neither plasma adrenocorticotropic hormone (ACTH) nor corticosterone was altered by the treatment conditions. This study corroborated previous reports that ionizing radiation exposure increased plasma levels of 8-hydroxy-2'-deoxyguanosine (8-OHDG), but no effect was observed in animals co-exposed to chronic noise. Plasma big-endothelin-1 (Big ET-1) was significantly reduced in animals exposed to a combination of noise and X-rays. The results indicated that (1) adaptation to chronic noise appeared to occur at the level of the hypothalamic pituitary adrenal (HPA) response, in spite of a compromise in overall body weight gain; and (2) ionizing radiation exposure might alter systems activated by stressor exposure and/or act independently to influence health outcomes.

Comet assay: rapid processing of multiple samples.

The present study describes modifications to the basic comet protocol that increase productivity and efficiency without sacrificing assay reliability. A simple technique is described for rapidly preparing up to 96 comet assay samples simultaneously. The sample preparation technique allows thin layers of agarose-embedded cells to be prepared in multiple wells attached to a flexible film of Gelbond, which improves the ease of manipulating and processing samples. To evaluate the effect of these modifications on assay sensitivity, dose-response curves are presented for DNA damage induced by exposure of TK6 cells to low concentrations of hydrogen peroxide (0-10 microM) and for exposure of human lymphocytes to X-irradiation (0-100 cGy). The limit of detection of DNA damage induced by hydrogen peroxide in TK6 cells was observed to be 1 uM for all parameters (tail ratio, tail moment, tail length and comet length) while the limit of detection of DNA damage in human lymphocytes was 10 cGy for tail and comet length parameters, but 50 cGy for tail ratio and tail moment parameters. These results are similar to those previously reported using the conventional alkaline comet assay. The application of SYBR Gold for detection of DNA damage was compared to that of propidium iodide. Measurements of matching samples for tail length and comet length were similar using both stains. However, comets stained with SYBR Gold persisted longer and were much brighter than those obtained with propidium iodide. SYBR Gold was found to be ideal for measuring tail length and comet length but, under present assay conditions, impractical for measuring tail ratio or tail moment due to saturation of staining in the head region of the comets.

Gluconeogenesis from glycine and serine in fasted normal and diabetic rats.

1. Non-anaesthetized normal and diabetic rats were fasted for 1 day, and [U-14C]glycine, or [U-14C]serine, or [U-14C]- plus [3-3H]-glucose was injected intra-arterially. The rates of synthesis de novo/irreversible disposal for glycine, serine and glucose, as well as the contribution of carbon atoms by the amino acids to plasma glucose, were calculated from the integrals of the specific-radioactivity-versus-time curves in plasma. 2. The concentrations of both glycine and serine in blood plasma were lower in diabetic than in fasted normal animals. 3. The rates of synthesis de novo/irreversible disposal of both amino acids tended to be lower in diabetic animals, but the decrease was statistically significant only for serine (14.3 compared with 10.5 mumol/min per kg). 4. Of the carbon atoms of plasma glucose, 2.9% arose from glycine in both fasted normal and diabetic rats, whereas 4.46% of glucose carbon originated from serine in fasted normal and 6.77% in diabetic rats. 5. As judged by their specific radioactivities, plasma serine and glycine exchange carbon atoms rapidly and extensively. 6. It was concluded that the turnover of glycine remains essentially unchanged, whereas that of serine is decreased in diabetic as compared with fasted normal rats. The plasma concentration of both amino acids was lower in diabetic rats. Both glycine and serine are glucogenic. In diabetic rats the contribution of carbon atoms from glycine to glucose increases in direct proportion to the increased glucose turnover, whereas the contribution by serine becomes also proportionally higher.

Gluconeogenesis from acetone in diabetic rats.

Previous investigations have demonstrated that acetone is a true, if minor precursor of glucose in vivo. In diabetic rats 1.30% of the carbon atoms of circulating glucose arises from acetone, whereas 0.67% does in normal 3-day fasted animals. Calculated from these fractions and the turnover rate of glucose, 48 micrograms/kg. min acetone-carbon is converted to glucose-carbon in diabetic and 16 micrograms/kg. min in normal rats. In both groups of rats the labelling of plasma lactate was stronger than that of glucose. In view of these results we conclude that: the transfer of C-atoms from acetone to glucose increases in diabetes; acetone remains a minor source of glucose even in ketonemic diabetic rats.

Gluconeogenesis from acetone in starved rats.

To non-anaesthetized rats starved for 3 days, [U-14C]acetone, NaH14CO3, L-[U-14C]lactate, [2-14C]acetate or D-[U-14C]- plus D-[3-3H]-glucose was injected intravenously. From the change in the plasma concentration of labelled acetone versus time after the injection, the metabolic clearance rate of acetone was calculated as 2.25 ml/min per kg body wt., and its rate of turnover as 0.74 mumol/min per kg. The extent and time course of the labelling of plasma glucose, lactate, urea and acetoacetate were followed and compared with those observed after the injection of labelled lactate, acetate and NaHCO3. The labelling of plasma lactate was rapid and extensive. Some 1.37% of the 14C atoms of circulating glucose originated from plasma acetone, compared with 44% originating from lactate. By deconvolution of the Unit Impulse Response Function of glucose, it was shown that the flux of C atoms from acetone to glucose reached a peak at about 100 min after injection of labelled acetone. In comparable experiments the transfer from lactate reached a peak at 14 min after the injection of labelled lactate. It was concluded that acetone is converted into lactate to a degree sufficient to account for the labelling of plasma glucose and is thus a true, albeit minor, substrate of glucose synthesis in starved rats.

Correction for metabolic exchange in the calculation of the rate of gluconeogenesis in rats.

The degree by which the rate of gluconeogenesis is underestimated when calculated from the transfer of 14C atoms from circulating precursors to plasma glucose was estimated from the ratio of the specific activity of plasma glucose and blood acetoacetate during the infusion of [2-14C]acetate in rats fasted for 3 days. The specific activity of blood acetoacetate was taken to be equal to that of its only precursor, the acetyl C atoms of hepatocellular AcCoA. These latter are assumed not to be available for the net synthesis of glucose and may be incorporated into the molecule by "metabolic exchange" in the hepatic oxalo-acetate pool only. By this approach 25% of the C atoms of plasma glucose have been estimated to arise from AcCoA. Multiplication by a correction factor H = 1.33 +/- 0.03 is being suggested to compensate for the metabolic exchange of 14C for 12C atoms when the rate of gluconeogenesis is calculated from the transfer of 14C atoms from labeled precursors to glucose.

Calculation of the rate of gluconeogenesis from the incorporation of 14C atoms from labelled bicarbonate or acetate.

The rate of gluconeogenesis in vivo may be estimated by the incorporation of 14C atoms from a labelled precursor into plasma glucose or by introducing 14C atoms into the pathway of gluconeogenesis at known stages by metabolites which in themselves do not contribute to the net synthesis of glucose (e.g., bicarbonate or acetate). The purpose of the investigation was to examine some of the assumptions involved in the calculation of gluconeogenic flux by the second approach. [2-14C]acetate or NaH14CO3 was infused to dogs, and the specific activity (SA) of glucose, bicarbonate CO2, urea, and lactate in the plasma was followed. The incorporation of 14C atoms from [2-14C]acetate into glucose allows the calculation of the degree of underestimation of glucose formation due to "metabolic exchange" in the hepatic oxaloacetate pool. The possible error introduced into this calculation by the incorporation of 14C atoms from 14CO2 (a product of acetate oxidation) was found to be negligible, but the heavy labelling of plasma lactate may possibly affect the estimate of metabolic exchange. It is proposed that in the calculation of the rate of gluconeogenesis from infused NaHCO3 the SA of hepatocellular and not of plasma bicarbonate CO2 should be related to that of plasma glucose. This latter is expected to equal the SA of plasma urea, since the sole precursor of its C atom is hepatocellular CO2. The rate of gluconeogenesis estimated from the SA(glucose)/SA(urea) ratio and a previously estimated correction factor for metabolic exchange was 51% of the glucose production in the postabsorptive state. The nearly identical SA(urea)/SA(CO2) ratios, irrespective of the tracer infused, indicated that plasma CO2 is a major precursor of urea C and that a large fraction of injected acetate is oxidized by extrahepatic tissues.