Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 6 uppsatser.

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In the fields of radiobiology and nuclear medicine there is considerable interest in the important role played by Auger electron cascades caused by inner-shell ionisation in realistic risk. It is necessary to quantify this risk when radionuclides are used on a routine basis as investigative, diagnostic and radiotherapeutic tools, whether the applications involve incorporated electron capture radionuclides or K-shell ionisation of selected stable nuclides by X-rays, as in "photon activation therapy". Relevant published survival data on biological damage caused by the internal emitters 125I, 77Br, 3H, 33P, 131I and 32P which are incorporated into the DNA of mammalian cells, bacteria (E. Coli) and bacteriophages have been collected and the results re-analysed in terms of the parameters of a new damage model to determine an inactivation cross-section for each internal emitter. These quality parameters are the absolute specification of radiation quality and are compared with cross-sections similarly determined for the effects of external radiations from heavy charged particles and photons (chapter 2). The inactivation probabilities obtained for the nuclides 125I, 77Br and 3H extend over a wide range of values depending on the type of nuclide and its distribution, the type of sensitive target and its shape and distribution, and the environmental temperature during both irradiation and post-irradiation incubation. The higher values approach those determined for heavy charged particles with the same mean free path for primary ionisation, and are an order of magnitude larger than would be expected for external irradiation with photon generated electrons. The results for 33P, 131I and 32P nuclides are appreciably smaller than that expected for external irradiation since the long range electrons dissipate most of their energy out of the sensitive target. A theoretical equation for X-ray production by accelerated electrons incident on a thick target has been revised by including factors to compensate for backscattering, direct and indirect ionisation, attenuation in the target and the incident angle of electrons (chapter 3). An electron accelerator X-ray machine capable of delivering monoenergetic photons up to ~ 4.8 gray/sec exposure dose rate from four different targets has been designed, constructed and tested (chapter 4) The biophysical mechanisms of direct and indirect radiation action has also been studied using the metallo-enzyme dihydroorotic dehydrogenase. The enzyme was irradiated both in dry state and in solution at different concentrations and at different dose rates using monoenergetic Cu-K photons from our X-ray machine. A technique was developed whereby it was possible to isolate and quantify each type of radiation action (chapter 5). The inactivation of the enzyme in both solution and in dry state was found to be a single-hit/single-target process. It was also found that in solution the inactivation of the enzyme was dose-rate-and concentration-dependent with efficiency of radical inactivation has an exponential dependence on dose-rate and the inverse of the enzyme concentration. A new model for the inactivation of the enzyme has been suggested and its parameters, namely direct and indirect cross-sections, geometrical cross-section, saturated concentration constant, root mean square diffusion constant, mean free path of radicals absorption, life time and G value of radical production, have been determined. It is expected that this model can be generalised to suit other enzymes (chapter 6).

Transitional cell carcinoma of the bladder is commonly encountered in urological practice. It affects people of a relatively young age causing economic and social distress to patients. In order to prevent the disease it is important to understand its pathogenesis. In this study, we have tumorigenically transformed a human urothelial cell by growing cells in a serum free, factor free, chemically defined culture. The tumorigenic property of the cell was determined by the generation of a tumor after inoculation into nude mouse. DNA fingerprinting analysis demonstrated the common back ground of the non-tumorigenic human urothelial cell and its tumorigenic transformant. This result also shows evidence of mutation occurring during transformation. By analysing conditioned medium, a significant reduction in the levels of soluble human stem cell factor and interleukin la were found in tumorigenic cell conditioned medium. A model derived from this evidence may suggest that tumor cells undergo further transformation under nutrient and growth factor deprived conditions. Intravesical chemotherapeutic agents in current use have shown moderate tumor-killing effects with some systemic or local side effects. Identification of a drug with better effect and less side effects is essential for the successful treatment of bladder tumors. Nitric oxide (NO) is a natural product of the human body with a role in tumor cell-killing. Thus by using NO as a chemotherapeutic agent we could at least expect limited side effects. Roussin's black salt (RBS) is a novel NO donor. Its cytotoxicity was tested on tumorigenic (T24) and non-tumorigenic (SV-HUC-1) human urothelial cells. The cytotoxicity of RBS was shown to be dose- and contact time- dependent. This cytotoxicity was enhanced by light irradiation and reduced in the presence of haemoglobin. The cytotoxic effect of RBS was also tested on CHO cells and the DNA repair deficient mutant xrs-5 cell line. Both colony forming and micronuclei forming assays demonstrated that xrs-5 cells are more sensitive to RBS than their counterpart. This result may indicate that NO is involved in the cytotoxicity of RBS and furthermore that DNA damage might be one possible mechanism by which the cytotoxic effect of RBS is expressed.

Transformation is a complex multistage process in vitro by which benign cells gradually acquire characteristics of tumour cells. Transformed C3H10T1/2 cells appear in vitro as multilayers of cells termed foci. Two main aspects of transformation of C3H10T1/2 cells in vitro have been investigated. Firstly, the quantitative assessment of the dose and dose-rate effects after irradiation with 250 kVp X-rays were examined and secondly the relationship between various properties of transformed cells in vitro and their tumourigenic potential in vivo. The induction of transformation was found to be linear with dose for 0.25 to 5 Gy X-rays. Lowering the rate at which the X-ray dose is delivered to the C3H10T1/2 cells lowers the observed transformation frequency by at least a factor of two. A variety of transformed phenotypes are observed in vitro and samples of these phenotypes were developed as cell lines and assessed for a number of properties. These properties were the ability to induce tumours in C3H mice and the ability to reconstruct foci in vitro. Other properties examined were growth in vitro parameters (lag time, doubling time and saturation density) as well as chromosome number and distribution. Tumour cell lines were also developed and assessed for the above properties. Transformation phenotypes induced by X-rays and alpha-particles were compared. Differences were found between some of the properties of the X-ray and alpha- particle induced transformants. In particular, higher proportions of X-ray induced transformants were tumourigenic while most of the alpha-particle induced transformants were non-tumourigenic and also tumours induced by the X-ray induced transformants appeared earlier and grew faster than the alpha-particle induced equivalent. The ability of the transformation phenotypes to reconstruct foci in vitro was greater for alpha-particle induced transformants (not including tumour cell lines) than for the X-ray induced transformants. The reverse was true for tumour cells where X-rays produced higher frequencies of reconstructed foci than alpha-particles. No differences were noted in in vitro growth parameters irrespective of transformation phenotype or radiation type apart from differences in saturation density where the transformation phenotypes (not including tumour cells) generally produced higher densities than the tumour cells for both X-rays and alpha-particles. Chromosome numbers in cells of the different transformation phenotypes (including tumour cells) induced by both X-rays and alpha-particles showed a greater spread and a general shift of the mean and modal chromosome number to lower values than that of untransformed C3H10T1/2 cells. The presence of metacentric chromosomes (Robertsonian chromosomes) was not unique to the radiation induced transformation phenotypes as most of the cell lines examined showed fewer of these chromosomes than the untransformed cells. The X-ray induced transformants (including tumour cells) generally produced more Robertsonian chromosomes than the alpha-particle equivalent. Correlation tests of the above properties with tumourigenicity of the transformed cells revealed a positive correlation of tumourigenicity with the ability to reconstruct foci. A negative correlation was noted between the ability to reconstruct foci and the mean and modal chromosome numbers.

It has been suggested that the proliferation of haematopoietic stem cells (CFU-S) in mice is controlled by the balance of inhibitory and stimulatory factors. In normal mice about 10 percent of the CFU-S population are in DNA synthesis. It has been suggested that a high concentration of inhibitor blocks CFU-S from entering into DNA synthesis. Following damage by cytotoxic agents such as drugs or irradiation about 30 - 50 percent of CFU-S were in DNA synthesis and also stimulator was shown to be present. In this study the entry of CFU-S into DNA synthesis following low and sub-lethal doses of whole body X-irradiation has been studied. Furthermore the stimulator producing cells were also characterized. The number of CFU-S in bone marrow was not affected following exposure to a dose of 0.5 Gy. However the number of committed progenitors for the granulocyte/macrophage lineage was significantly reduced. The percentage of CFU-S in DNA synthesis was found to increase to 37.0+/- 7.0 percent at 30 minutes and 43.9+/-11.2 percent at 2 hours from that observed in unirradiated mice. However at 6 hours the percentage was 14.8 8.1 percent. At a sub-lethal dose of 4.5 Gy, the percentage of CFU-S in DNA synthesis increased to 34.0+/-14.0 percent at 6 hours after exposure, however before this time the percentage remained at a similar level to unirradiated control mice. When plugs of bone marrow were irradiated in-vitro at 0.5 Gy and 4.5 Gy doses, the time of CFU-S entering into DNA synthesis was the same as following in-vivo irradiation. The dose response curve of CFU-S entering into DNA synthesis when measured at 2 hours after exposure showed that the percentage was increased as the dose was increased and reached 30-50 percent at a dose of 0.5 Gy. Above this dose the CFU-S population was not stimulated at this time. When the percentage of CFU-S in DNA synthesis was measured at 6 hours after exposure, the values were the same as control for doses less than 0.5 Gy and above this dose the values were 30-50 percent. The presence of stimulator in bone marrow after irradiation was found to parallel the proliferative activity of CFU-S. The CFU-S population obtained 1 hour after 1.5 Gy was shown not' to respond to stimulator as CFU-S from normal bone marrow dia. The conditioned media prepared from bone marrow of mice Irradiated at 9.0 Gy (1 to 5 days post Irradiation) increased the proportion of CFU-S from normal bone marrow in DNA synthesis to 30-50 percent. The depletion of Thy1.2+ cells from regenerating bone marrow did not affect the ability to produce stimulator. However when Fc+ and Ia-2k+ cells were removed the stimulator production was affected. This suggests that the stimulator producing cells were radioresistant, Thy1.2-, Fc+ and Ia-2k+.

An extensive survey made of published survival data of damage by ionizing radiation to mammalian cells in vitro has led to the new conclusion that the damage is determined by the specific ionization or the mean free path between ionizing events along the charged particle tracks. The optimum damage is observed when the mean free path is equivalent to the DNA double strand spacing of 1.8 nm. Therefore, the biological mechanism of ionizing radiation to mammalian cells in vitro is intra track dominant. A 100 keV electron accelerator has been constructed and commissioned to produce a broad beam irradiation field of greater than 1 cm diameter. The fluence rate may be adjusted from 10⁸ cm-2-sec-1 downwards to enable further development as a chronic irradiation facility. Another new feature of the accelerator is that it incorporates a differential vacuum system which permits irradiation of the monolayer cell cultures to be carried out in normal pressure. Experiments of irradiation to Chinese hamster cells, by 241Am alpha particles at low fluence rate, have supplied satisfactory data for testing a new DNA-rupture model which is under development. For V79 cells irradiated at a low fluence rate of 10⁵ cm-2-min-1, when survival data were fitted into the model, new biophysical parameters were extracted and a proposal was made that the repair phenomenon of cellular survival at very low doses is determined by three time factors; the irradiation time, the damage fixation time and the repair time. The values obtained were 3-4 hours for the mean repair time, and more than 10 hours for the damage to be considered permanent. Details of the monolayer cell culture technique developed and used in the present experiments are described. Consideration has been given to the significance of the results obtained from the study in radiation protection and in radiotherapy. In future studies it is recommended that more attention should be paid to measure the ionization events of the radiation studied. Towards the objective of producing a unified dosimeter, more studies are needed to correlate the results for electrons and neutrons for which less data are available. In this thesis the background and the basic theories are introduced in Chapters I and II. Experimental details are described in Chapter III on physical aspects and Chapter IV on biological aspects. Finally the results and discussion are presented in Chapter V.

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Workshop on X-rays from electron beams with special emphasis on possible developments at ELBE

In this study the direct effect of ionising radiation on DNA plus additives has been studied using both ESR spectroscopy and plasmid DMA (for strand break analysis). The primary radicals were identified as the thymine radical-anion, T', and guanine radical-cation, G'*'. Under normal conditions these were formed in approximately equal yields as defined by careful computer simulations. Certain additives such as oxygen, nitroimidazoles, silver ions and the rest of the nuclear complement (i.e. RNA and histone proteins) , were added to study their effects on the relative yields of T" and G". In all cases, they were shown to capture electrons in competition with T" and have little or no effect on the yield of G"*". In the case of oxygen and nitroimidazoles the effect of reducing the yield of T" radicals was looked at using strand break analyses. Essentially this was found to protect the DNA. Since both single and double strand breaks were found at significant levels when G+ and T~ were the only detectable initial radicals, one must conclude that these radicals are responsible for strand breaks. Fran the relatively high number of double strand breaks found, we deduce that G'*' and T" centres must be close togetlier (in a range of ca. 10-50 A), and that both may give rise to strand breaks, by as yet undefined pathways. In a separate study (Chapter 4), the reaction between superoxide ions, O2"/ and dimethyl formamide has been investigated by ESR spectroscopy. Strong evidence in favour of addition of O2" at the C=0 group to give a relatively stable peroxy radical intermediate has been obtained. This has implications for the mechanism of action of O2" formed both as a result of radiation damage and by other means. Appendix I describes a study of various simple aldehyde and ketone radical-cations, using ESR spectroscopy. Interpretations of these spectra are given, together with structural implications. Appendix II is a paper on work carried out on the ESR spectra of hydroxyl radicals in aqueous glasses. This work was done in collaboration with H. Riederer and J. Hiittermann.