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This study examined whether radiation sensitivity measured by lymphocyte apoptosis could be ameliorated by a complex anti-oxidant/anti-ageing dietary supplement. We also examined lymphocytes from both genders of normal (Nr) mice as well as transgenic growth hormone (Tg) mice that express strongly elevated reactive oxygen species processes and a progeroid syndrome of accelerated ageing. We introduce Tg mice as a potentially valuable new model to study radiation sensitivity. Isolated lymphocytes from all experimental groups were exposed to gamma radiation and the time course of apoptosis was measured in vitro. Kinetics of radiation-induced apoptosis was similar among groups, which peaked at 8 h, but maximal levels differed significantly between groups. Nr male mice had 60% lower levels of radiation-induced apoptosis than Tg males, supporting our hypothesis that Tg mice would be radiation sensitive. The dietary supplement protected lymphocytes in male mice of both strains, with proportionally greater reductions in Tg mice. Lymphocytes from female mice (both Nr and Tg) were highly radiation resistant compared to males and the supplement provided no additional benefit at the doses used in this study. These results highlight that radiation-induced apoptosis is complex and is modified by genotype, dietary supplements and gender.

The relative biological effectiveness (RBE) of neutrons varies from unity to greater than ten depending upon neutron energy and the biological endpoint measured. In our study, we examined apoptosis in human lymphocytes to assess the RBE of low energy 280 keV neutrons compared to Cs gamma radiation and found the RBE to be approximately one. Similar results have been observed for high energy neutrons using the same endpoint. As apoptosis is a major process that influences the consequences of radiation exposure, our results indicate that biological effect and the corresponding weighting factors for 280 keV neutrons may be lower in some cell types and tissues.

Purpose: To develop an artificial neural network (ANN) model of apoptotic response in gamma irradiated human lymphocytes. To assess the feasibility of training ANN radiobiological models using data collected with flow cytometry., Materials and Methods: Irradiated isolated human lymphocytes were labelled with Annexin V-Fluorescein Isothiocyanate (FITC) and 7-Amino-Actinomycin D (7AAD) then analysed using flow cytometry. Twenty-four dose responses per donor from 14 donors were collected from a flow cytometer and used in model development as the training and cross-validation datasets. The general ANN model architecture was a multi-layer perceptron using the mean squared error of a cross validation dataset as the objective function. The ANN model was optimized by varying the number of hidden layers and the number of processing elements per layer. The optimized model constituted of three hidden layers with 80, 40, and 10 hidden layers in the first, second, and third layers respectively., Results: The optimized model was used to simulate dose responses at the training doses of 0, 2, 4 and 8 Gray. A strong agreement between the model and measured dose responses was observed. The model was also used to simulate a dose response at 0.1 Gray and results were compared to the measured dose response from a donor not used in model development. Again, strong agreement between the model and the observed dose response was found., Conclusions: This study shows that artificial neural networks can be trained to provide high resolution, high accuracy models of multivariate radiobiological data collected by flow cytometry.

We have compared dose-rate effects for gamma-radiation-induced apoptosis and micronucleus formation in human lymphocytes. Long-term assessment of individual radiation-induced apoptosis showed little intraindividual variation but significant interindividual variation. The effectiveness of radiation exposure to cause apoptosis or micronucleus formation was reduced by low-dose-rate exposures, but the reduction was apparent at different dose rates for these two end points. Micronucleus formation showed a dose-rate effect when the dose rate was lowered to 0.29 cGy/min, but there was no accompanying cell cycle delay. A further increase in the dose-rate effect was seen at 0.15 cGy/min, but was now accompanied by cell cycle delay. There was no dose-rate effect for the induction of apoptosis until the dose rate was reduced to 0.15 cGy/min, indicating that the mechanisms or signals for processing radiation-induced lesions for these two end points must be different at least in part. There appear to be two mechanisms that contribute to the dose-rate effect for micronucleus formation. One of these does not affect binucleate cell frequency and occurs at dose rates higher than that required to produce a dose-rate effect for apoptosis, and one affects binucleate cell frequency, induced only at the very low dose rate which coincidentally produces a dose-rate effect for apoptosis. Since the dose rate at which cells showed reduced apoptosis as well as a further reduction in micronucleus formation was very low, we conclude that the processing of the radiation-induced lesions that induce apoptosis, and some micronuclei, is very slow in quiescent and PHA-stimulated lymphocytes, respectively.

We have investigated heat- and radiation-induced apoptosis in human lymphocytes in vitro. We have previously shown that apoptosis was induced by radiation at doses as low as 0.05 Gy. Here we report that heat induced apoptosis in human lymphocytes in a temperature- and time-dependent manner. Temperatures at or below 42 degrees C, for up to 90 min, did not cause lymphocytes to undergo apoptosis, whereas temperatures at or above 43 degrees C, for 30 min and longer, did induce apoptosis. Lymphocytes were protected against apoptosis induced by 44 degrees C heat by a prior heat shock of 42 degrees C for 30 min. Heat-induced thermal tolerance developed immediately following the inducing heat shock, was greater after 4 h, and persisted for at least 24 h. While heat also induced radiation resistance, this change was minor and not apparent until about 24 h after the heat shock. Prior to the development of radiation resistance, heat shock sensitized lymphocytes to radiation-induced apoptosis. We have previously shown that radiation-induced apoptosis in lymphocytes varies between donors and therefore may be useful in assessing individual radiosensitivity. We report here that heat also induced variable levels of apoptosis in lymphocytes from different donors, although the range of responses was not as large as those observed with radiation-induced apoptosis. In summary, heat shock induces tolerance to heat-induced apoptosis and results first in sensitization and then protection of lymphocytes against radiation-induced apoptosis.

We have tested the possibility of using apoptosis (programmed cell death) in human peripheral blood lymphocytes as a short-term biological dosimeter. Lymphocytes isolated from whole blood were irradiated in culture with 250 kVp x-rays or 60Co gamma rays. Two assays were used to measure apoptosis in lymphocytes after irradiation: in situ terminal deoxynucleotidyl transferase assay and fluorescence analysis of DNA unwinding assay. Similar qualitative and quantitative results were produced by the assays, supporting the notion that the fluorescence analysis of DNA unwinding assay measured DNA fragmentation associated with apoptosis. Induction of apoptosis in lymphocytes irradiated in vitro was proportional to dose and could be detected following exposures as low as 0.05 Gy. Lymphocytes from individual donors had reproducible dose responses. There was, however, variation between donors. X-ray and gamma-ray exposures induced similar levels of apoptosis at similar doses. The induction kinetics of apoptosis in vitro indicate a maximum is reached about 72 h after irradiation. In conclusion, the in vitro experimental evidence indicates that radiation-induced apoptosis in human lymphocytes has the kinetics, sensitivity, and reproducibility to be a potential biological dosimeter.

We have investigated the influence of the cellular adaptive response to ionizing radiation on radiation-induced apoptosis in human cells. The adaptive response is believed to be a protective mechanism that confers resistance to the detrimental effects of ionizing radiation and that can be induced by different agents, including hyperthermia and radiation. We have used fluorescence analysis of DNA unwinding (FADU) to assay the induction of apoptosis in human peripheral blood lymphocytes by ionizing radiation. Using the FADU assay, we have observed the initial radiation-induced DNA damage, its subsequent disappearance due to enzymatic repair, and its time- and dose-dependent reappearance. We believe this reappearance of DNA damage to be indicative of the DNA fragmentation event associated with apoptosis. This interpretation has been supported at the individual cell level using an in situ terminal deoxynucleotidyl transferase (TDT) assay (Apoptag, Oncor Inc.), which detects the 3'-hydroxyl ends of fragmented DNA, and by fluorescence analysis of nuclear morphology in Hoechst 33258 stained cells. Pretreatment of cells with low-dose gamma-radiation (0.1 Gy) or mild hyperthermia (40 degrees C for 30 min) altered the extent of radiation-induced (3 Gy) apoptosis. Both pretreatments sensitized lymphocytes to become apoptotic after the 3-Gy radiation exposure. This sensitization may represent an adaptive response mechanism that reduces the risk that genetically damaged cells will proliferate. The ability to modify the probability of radiation-induced apoptosis may lower the cancer risk from a radiation exposure.