Browsing by Author "Blekkenhorst, Gerhardus Hendrikus"

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    The analysis of radiation-induced micronuclei in peripheral blood lymphocytes for purpose of biological dosimetry
    (1995) Le Roux, Jacques; Blekkenhorst, Gerhardus Hendrikus
    In the investigation of radiation accidents, it is of great importance to estimate the dose absorbed by exposed persons in order to plan their therapy. Although occasionally in these situations physical dose measurements are possible, most often biological methods are required for dose estimation. The aim of this investigation was to assess the suitability of the cytokinesis blocked (CB) micronucleus assay as a biodosimetric method using lymphocytes irradiated in vivo. The approach adopted to achieve this was to estimate whole body doses by relating micronuclei yields in patients undergoing radiotherapy treatment with an in vitro radiation dose-response curve. These biologically derived estimates were then compared with the corresponding doses obtained by physical measurement and calculation. As a first approach a study was performed of the in vitro dose-response of gamma-ray induced micronuclei following cytokinesis-block in the lymphocytes of peripheral blood samples obtained from 4 healthy donors. The results indicated that the distribution of the induced micronuclei were overdispersed. Furthermore, a linear dose-response relationship was established when a curve was fitted to the data by an iteratively reweighted least squares method. By means of an analysis of covariance it was demonstrated that this result is in agreement with the dose-response relationships found by various other workers (Fenech et al., 1985; Fenech et al., 1986; Fenech et al., 1989; Balasem et al., 1992, and Slabbert, 1993). To assess the suitability and accuracy of dose assessment using the CB micronucleus assay for in vivo exposure of lymphocytes, blood samples obtained from 8 patients undergoing radiotherapy before, during and after treatment were examined. The physical doses of these patients were determined according to conventional radiation treatment plans and cumulative dose-volume histograms. The dose-volume histograms permitted calculation of integral doses and subsequently the estimate of equivalent whole-body doses. The results of the CB micronucleus assay applied to peripheral blood lymphocytes of 6 patients undergoing fractionated partial-body irradiation showed a dose-related increase in micronucleus frequency in each of the patients studied. This demonstrated that micronuclei analysis may serve as a quantitative biological measure of such exposures. The pooled data of these patients compared to the pooled data of the healthy donors show that there was no statistically significant difference between in vitro and in vivo results, however a slightly lower induced micronuclei frequency was observed after in vivo exposure. When the biological dose estimates for equivalent whole-body doses obtained from the in vitro dose response curve were compared with calculated physical doses, it was found that: biologically estimated dose = 0.936 physical dose. However, there was inadequate statistical evidence to discard the hypothesis that the gradient of the equation was equal to one. Therefore, the analysis of micronuclei induced in lymphocytes in vivo yields highly quantitative information on the equivalent whole-body dose. The negative binomial method was used for analysing the micronucleus data from two patients who received single, relatively larger tumour doses of 10 Gy each, with the objective to obtain estimates of the exposed body fraction and the dose to this fraction. The dose estimates to the irradiated volume were found to be within 30% of the physical tumour dose. The irradiated volume estimates seemed to be higher than the physically calculated volumes but by discarding the correction for the loss of cells due to interphase death the agreement was good between the physically and biologically determined integral doses. This study has revealed that the CB micronucleus assay appears to offer a reliable, consistent and relatively rapid biological method of whole body dose estimation. It is recognised that further corroborative work using the techniques described in this thesis is required for estimating localized exposure.
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    Continuous low dose rate irradiation of the rat brain
    (1999) Madhoo, Jitesh; Blekkenhorst, Gerhardus Hendrikus
    The reported median survival time for patients who are diagnosed with high grade astrocytomas and who undergo postoperative radiotherapy is of the order of 24 to 40 weeks. The course of radiotherapy administered to these patients takes up a considerable portion of their expected survival time. Therefore, any means of reducing the treatment time may contribute to an enhanced quality of life for these patients. A potentially useful method for the reduction of the treatment time may be achieved with the use of continuous low dose rate external beam radiotherapy, where the treatment is administered over a 12 to 24 hour period. A relationship between fractionated and continuous low dose rate irradiation has been reported for skin, however, no such relationship has been reported for the brain. Low dose rate protocols that are equivalent in effect to fractionated (conventional) protocols can be derived using the linear quadratic theory, provided that quantitative radiobiological data for normal tissue (brain) is known. Thus, the aim of the current study is to test the radiation tolerance of the rat brain to low dose rate and fractionated radiation in order to establish the values for the parameters of the linear quadratic model.
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    The effect of cis-platinum alone or in combination with radiation on mouse lung
    (1999) Duffett, Rodger Vincent; Blekkenhorst, Gerhardus Hendrikus; Abratt, Raymond
    Cis-platinum is a widely used cytotoxic agent with known radiosensitising properties. It is used in the treatment of various types of lung cancer that may include radiation to the lung as part of the treatment protocol. There is little evidence and some conflict as to whether it sensitises pulmonary tissue to the effects of radiation treatment. This project investigates the effect of cis-platinum alone or in combination with radiation on mouse lung. Four end points were used to evaluate treatments. They were: the release of pulmonary surfactant, changes in breathing rate, a histology based score of damage and changes in TGF-β - a cytokine important in the development of fibrosis. Single doses of either cis-platinum or radiation, cis-platinum given immediately before a single dose of radiation, cis-platinum given immediately before the first of two fractions of radiation and cis-platinum given at various times before and after a single dose of radiation were investigated. Cis-platinum alone was observed to cause an increase in the phospholipid content of lavaged surfactant. Cis-platinum was observed to cause an early release in surfactant and a trend existed for it to induce an early increase in breathing rates as compared to that induced by radiation alone. Cis-platinum was observed to increase radiation damage as assessed using a histology based scoring system. Higher TGF-β levels in lavaged surfactant were observed in C57 /Bl mice as compared to Balb/C. No difference in TGF-β levels was seen in homogenised lung between the strains. Cis-platinum may cause changes in TGF-β in C57/Bl mice but further work is necessary to confirm this.
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    The effects of the modification of energy metabolism on cellular response to ionizing radiation
    (1997) Hunter, Alistair John; Blekkenhorst, Gerhardus Hendrikus
    It is generally accepted that energy is required for repair of radiation-induced damage in living cells. Some of this energy is probably provided by adenosine triphosphate (ATP), which is derived from energy substrates via energy metabolism. This dissertation follows two general avenues. The first explores the effect of radiation on ATP levels after irradiation of cells. The second investigates the effect of inhibitors of certain pathways associated with energy metabolism on radiation response. It was proposed that ATP levels might be raised after irradiation in some systems and that this rise in ATP might be due to compensatory mechanisms related to repair. Experiments were conducted using B16 melanoma cells in vitro and using normal murine liver and CaNT tumours in vivo. ATP concentration was measured in extracts of these cells after irradiation using the luciferase-luciferin method. No major changes from unirradiated controls were found. Several types of substrates exist from which cells can derive energy, including glucose and glutamine which are initially metabolised via glycolysis and glutaminolysis, respectively, before their products are further metabolised in respiration. Since energy is necessary for repair of radiation damage, it has been proposed that the inhibition of energy metabolism might alter the radiation response of cells. An inhibitor of glycolysis, 2-deoxyglucose (2DG), and an inhibitor of glutaminolysis, aminooxyacetic acid (AOA), were administered to CHO cells in vitro to determine the effects of these substances on cellular radiosensitivity and repair. Repair was assessed by means of a split radiation dose experiment. The design of such an experiment required that cells be exposed to inhibitory test media for different times between two fractions of radiation. Any changes in clonogenic survival with time between tween fractions could, therefore be as a result of repair effects or as a result of changes in radiosensitivity. A method of estimating and subtracting the effects of radiosensitivity to make conclusions concerning repair is presented and discussed. Most combinations of 2DG, AOA, glucose omission and glutamine omission in culture media resulted in reductions in repair rate but the extent of repair was found to vary from one medium variation to the next. In addition, the effects of various culture media on glycolysis/PPP (glycolysis/pentose phosphate pathway) and glutaminolysis were investigated by determining the production of CO2 and lactate from radiolabelled-glucose and -glutamine substrates. It was apparent that the presence of either of the inhibitors, 2DG or AOA, could inhibit the activity of glutaminolysis and reduce oxygen consumption. 2DG was shown to inhibit glycolysis/PPP but AOA was shown to stimulate glycolysis/PPP, suggesting a regulatory link between glutaminolysis and glycolysis/PPP. The presence of either inhibitor resulted in a reduction in the rate of radiation damage repair. The medium which had the most significant effect in respect of repair inhibition and increased radiosensitivity was medium lacking both glucose and glutamine and containing both 2DG and AOA. This medium was shown to inhibit oxygen consumption and to result in a depression of both cellular glycolysis/PPP and glutaminolysis. The effect of 2DG on the rate of growth and radiation induced growth delay of three murine tumours in vivo was assessed. 2DG alone inhibited the growth of B16 tumours. However, 2DG alone produced little if any change in the rates of growth of Fib/T tumours and rhabdomyosarcomas but the combination of 2DG and AOA produced an inhibition of growth in the Fib/T tumour. 2DG appeared to enhance the effects of radiation in the Fib/T and B16 tumours but not in the rhabdomyosarcoma, although, in the Fib/T, the combination of AOA, 2DG and radiation was less effective in inhibiting tumour growth than was radiation alone. The effects of radiation and 2DG did not appear to be additive in the Fib/T tumour and the B16 tumours which may imply an influence of 2DG on repair or radiosensitivity. This work suggests that the effects of radiation can be altered by manipulation of metabolic pathways associated with the supply of energy. However, a complex interaction of pathways is probably also involved and it is the detail of this interaction which may partially determine the severity of radiation response.
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    Inactivation synergy between high energy neutrons and ⁶⁰Co gamma rays
    (1993) Slabbert, Jacobus Petrus; Blekkenhorst, Gerhardus Hendrikus
    The interaction between sublesions produced by neutrons [p(66)/Be and d(16)/Be] and ⁶⁰Co γ-rays was investigated using mammalian cells, meristematic cells and human lymphocytes. The quality of each radiation source was quantified in terms of molecular yield per unit dose absorbed in a ferrous sulphate xylenol orange solution and was found to vary inversely with the mean LET of the radiation field. Inactivation parameters determined for mammalian and meristematic cells were not significantly different following simultaneous or sequential exposures to d(16)/Be neutrons and ⁶⁰Co γ-rays. Synergistic interaction was observed to be most pronounced in a radiation mixture consisting of about one part neutrons and three parts photons and appeared to be optimal at approximately 5 Gy. This phenomenon led to dose enhancement ratios that increase with radioresistance. Multi-target parameters indicated that on a per gray basis, priming doses of p(66)/Be neutrons and ⁶⁰Co γ-rays induce comparative levels of sublethal damage. However, non-parametric analysis of the survival data showed that mammalian cells regard a priming dose of neutrons as somewhat less effective than an iso-effective photon dose. A greater measure of synergy was observed between photons and priming doses of neutrons with less build-up. This is however mainly due to higher levels of biological damage induced with a more potent configuration of secondary charged particles. Interaction factors compared at levels of iso-effect tend to be smaller when the LET of the priming dose was increased. Split-doses of neutrons in the absence of build-up resulted in "negative" repair. The validity of proposed biophysical models was tested using meristematic cells, as the response of these cells show an apparent absence of intertrack damage. Contrary to expectations, synergistic interaction was observed for both growth delay measurements and micronuclear formations. Chromosome aberrations showed synergy between neutron and photon damage in human lymphocytes, as predicted by interaction functi ons. However, the synergistic interaction noted with micronuclear formation in binucleate cells was at variance with predictions based on biophysical models.
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    The potential of the superoxide dismutase inhibitor, diethyldithiocarbamate as an adjuvant to radiotherapy
    (1990) Kent, Charles; Blekkenhorst, Gerhardus Hendrikus
    It has long been known that oxygen has the potential to be toxic to biologic systems and that this toxicity is not due to oxygen itself, but due to the production of oxygen radicals. One of these potentially toxic radicals, superoxide (O₂⁻) can be generated as a result of ionizing radiation, and if not adequately removed can proceed to cause cell damage. Superoxide dismutase (SOD) is one of the key enzymes involved in the defence against oxygen toxicity. SOD activity can be inhibited by diethyldithiocarbamate (DOC), a powerful copper chelator. If inhibition of SOD by DOC increases the lifetime and effectiveness of radiation induced O₂⁻, it follows that the potential exists for DOC to enhance the effect of radiation. DOC is however also a thiol compound, and thus may act as a radioprotector by modifying tissue oxygenation status or by free radical scavenging. This study has concerned itself primarily with the inhibition of superoxide dismutase by diethyldithiocarbamate in order to sensitize tumours to ionizing radiation. The use of DOC as an inhibitor of SOD has however meant that any sensitization resulting from SOD inhibition could be masked by a radioprotective effect by DOC. The inhibition of SOD by DDC was confirmed in a murine rhabdomyosarcoma, and it was shown that this inhibition can be maintained for up to twenty-four hours after DDC administration. It was hypothesised that there was a potential for the radioprotective effect of DDC to be overcome, if the levels of DDC were low enough at the time of irradiation. Indeed, if DDC was removed from the growth medium of B16 mouse melanoma cells in culture prior to irradiation, a significant sensitization was demonstrated. It was shown that DDC could act as both a radiosensitizer and as a radioprotector in the same experiment. The dominant action of DDC was found to be dependent on the time allowed between DDC administration and irradiation. If this time was approximately 4 hours, it was possible to show a radiosensitizing effect by means of a tumour growth delay assay. This time modulation effect of DOC was shown in larger tumours, rather than smaller tumours, which could indicate that tumour oxygenation is an important criterion in determining the response to radiation of DOC treated cells. It was shown that B16 mouse melanoma cells exposed to 43°C after DDC pre-treatment were sensitized to thermal damage. This work suggests that some caution should be exercised when DDC is put forward as either a radiosensitizer or a radioprotector in the clinic, but that DDC may have potential as a thermosensitizer.
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