Radiation Biophysics – Overview (Research)
Exchanges with the local Teachings Hospitals and pursuing Radiobiology matters that is of direct clinical relevance is an ongoing project. As a result 4 research papers on the clinical outcome of particle therapy protocols have been published during recent years. This includes a retrospective study evaluating the control rates of skull base meningiomas for patients treated using stereotactic proton therapy. By analysing different dose / fractionating schedules a tissue specific repair constant could be estimated from the clinical data that proves to be most useful to calculate the physical radiation dose needed for a given treatment protocol (Int. J Rad. Oncol. Biol. Phys. 2001). Also, it could be demonstrated that the hypofractionated proton treatments given at iThemba is iso-effectiveness to both conventional fractionated protocols and radiosurgery treatments given at other facilities.
Synopsis of Research Projects Performed in the Cellular Radiobiology Laboratory in Collaboration with Outside Users.
The RBE of the 200 MeV proton beam for late lung tolerance. (J. Gueulette, B de Coster, J Slabbert, L Bohm).
Late effects for fractionated irradiations (1,3 and 10 fractions) were used to derive a/b values for different positions in the SOBP of the clinical proton beam. The repair characteristics were found to diminish with time and low values (2 – 4 Gy ) were finally obtained. This in expectation for late responding tissue. RBE values however did not vary with fractionation.
Micronuclei induction in an ovarian cell type by cisplatin and 5-fluorouracil using immunospecific microspheres. (A Santos, J P Slabbert, E Truter)
These chemotherapeutic agents were encapsulated in antibody-coated microspheres and administered to an ovarian cancer cell line. Cisplatin by itself had no outcome but the two drugs together were observed to produce a synergistic effect.
Radiotoxicity of [123I] 4-Iodoantipyrine in normal and cancer cells. (B Smit, J P Slabbert, J Langenhoven)
Auger electrons from the decay of 123-Iodine carried across the cell and nuclear membrane when labelled to the organic compound induced very high levels of micronuclei in both an ovarian cancer cell type and normal epithelial cells. Differences noted were corresponding to variations in inherent radiosensitivity.
In the first part of a study to compare the potential therapeutic gain of different energy neutrons the data for the p(66)/Be beam have been generated for a panel of 14 different cancer cell types. A significant correlation between variations in neutron sensitivity and photon sensitivities was observed.
Live cell sorting using flow cytometery. (J Michie, C Muller, J P Slabbert, L Skarsgard)
Live cell sorting has been attempted in order to seed cell numbers with high accuracy so as to be able to estimate the sub-structure at low doses of the biological response to protons. Although runs with human lymphocytes were successful, even spectra for other cells types in culture were most variable and accurate cell seeding could not be obtained.
RBE of the NAC 200 MeV proton beam for inactivation of jejunal crypt cells following fractionated irradiations. (J. Gueulette, B de Coster, J P Slabbert, L Bohm).
Repair characteristic for early tissue response to protons given in 1, 3, and 10 fractions were quantified in a 7 cm SOBP. RBE in distal part of SOBP considerably higher than at other depths. a/b values of 7 – 8 Gy calculate from data and is consistent with that of early responding tissue.
Radiosensitivity of Neuronal cells (J Akudugu, J P Slabbert, A Serafin, L Bohm)
A comparison was made between neuroblastoma cells and peripheral lymphocytes of susceptibility to radiation induced micronuclei. The more radioresistant neuroblastoma cells show higher survival but paradoxically formed more micronuclei than lymphocytes.
Variations in the radiosensitivity of human tumour cells to NAC neutrons and cobalt-60 gamma rays. (J P Slabbert, T Theron, A Serafin, D Jones, L Bohm, F Zolzer, C Streffer)
Extended measurements were performed on the response of different cancer cell types to neutrons and photons to estimate the potential for therapeutic gain. A strong correlation between cellular response to the two treatment modalities were observed with the result that RBEo values only reduce on average with radioresistance and as such no significant measure of therapeutic gain could be observed.
The influence of p53 status on radiation-adaptive response was investigated in a wild-type cell line and one that has mutant inactive p53 status. Apoptosis adaptive response was induced in both cell types. Adaptive response with respect to micronuclei however was only observed in the p53 mutant cells.
Influence of a strong magnetic field on the radiobiological properties of a high energy X-ray beam. (H Burger, A van Rensburg, J Slabbert)
The track structure of secondary charged particles by 8 MV X-rays is expected to be distorted when exposed to a 1 Tesla magnetic field. Biological evidence for a radiation field with a possible higher ionisation density was investigated to determine possible use in clinical radiotherapy. The magnetic field resulted in higher levels of biological damage per unit dose for lymphocytes exposed to these X-rays, but dispersion parameters of micronuclei in the cells did not concur with what is expected with an increase in ionisation density.
Radiation Induction of micronuclei by neutrons in neuronal cells with different photon sensitivity. (J Akudugu, J P Slabbert, L Bohm)
Neutron sensitivities of a panel of 13 neuronal cells proved to vary widely to the clinical neutron beam and as well as to photons and micronuclei frequencies for different cell types treated with these modalities are related. It was found that the micronuclei frequency was much higher in some radioresistant cell lines. In a subset of some cell types no correlation between micronuclei formation and radiosensitivity could be established. It was concluded that it would be simplistic to translate micronuclei frequency into radiosensitivity
Effect of Cis-Diamminodichloroplatinum and [Rh(fctfa(coD)] on aerobic and hypoxic cells irradiated with neutrons. (N Falzone, C Medlen J P Slabbert, D Jones)
Cisplatin and a novel rhodium complex have been tested for radiosensitisation properties using 8 MV X-rays and neutrons. Dose modifying factors for photons were somewhat larger than that for neutrons and the two drugs show preferential sensitisation for cells treated under hypoxic conditions.
The study was performed to establish if radioprotection by cyteamine is significant for cells irradiated with the neutron therapy beam. This as neutron therapy for children has been planned and the usage of the radioprotector amifostine was suggested to protect normal tissue. A dose modifying factor of 2.1 was measured with neutrons and this is considerably less than that seen with photon irradiations, DMF ~ 3.4. It was concluded that amino – sulphydral compounds do however offer a considerable degree of radioprotection to cells irradiated with fast neutrons, notwithstanding the fact that indirect actions are much less important with high-LET treatment modalities. Normal tissue response is therefore likely to benefit from using such drugs during neutron therapy.
Radiobiology at the single cell level: Technical developments. (C Michelet J P Slabbert, W Przybylowicz, Ph Moretto).
In preparation for a single ion beam / single cell irradiation facility developed in Bordeaux France, test measurements were conducted using a computer controlled microscope stage to follow the fate of individual cells to radiation. The viability of live cells was tested using a Hoechst stain. The latter prove to be non-toxic and a suitable fluorescent agent to map cell coordinates. CHO-K1 cells exposed to priming doses of 0.1 Gy and then treated with a higher challenging dose, failed to show an adaptive response and as such is not suitable for use in single cell irradiation experiments.
Response of different human tumour cells to low and high-energy neutrons. (J P Slabbert, T Theron, F Zölzer, C Streffer and L Böhm).
The potential for therapeutic gain from using the high-energy neutron beam at the NAC has been investigated repeatedly in the past. From this it became clear that the variation in neutron sensitivities for cells exposed to p(66)/Be neutrons was much more than anticipated and that the therapeutic gain from using neutrons is limited to only a few cell types that proved to be very resistant to 60Co gamma rays but being relatively sensitive to neutrons. In this work cellular response is quantified using an appropriate clonogenic assay and the response of cells to X-rays or cobalt-60 gamma rays are compared to that in a d(14)/Be neutron field (Essen, Germany) and to that in a p(66)/Be neutron beam (Faure).
Results obtained with this series of measurements confirm our previous observations with the NAC neutron beam indicating that large variations in neutron sensitivities are evident and that this is strongly correlated to that seen with photons. As a result the RBE for NAC neutrons does not increase in a significant manner and therapeutic gain is limited to a few very radioresistance cancer cell types. By contrast, the response of different histological cell types irradiated with d(14)/Be neutrons show much less variation in response, are less related to the response of the same cells exposed to X-rays and demonstrates a much higher likelihood in obtaining therapeutic gain.
Recoil proton, alpha particle and heavy ion impacts on microdosimetry and RBE of fast neutrons: analysis of kerma spectra calculated by Monte Carlo simulation (J-P Pignol, J P Slabbert)
The FLUKA and MCNP Monte Carlo codes were used to simulate the total proton and heavy ion kerma spectra for the clinical neutron beam. From this the changes in RBE could be linked to both slow energy protons and alpha particles yielded by (n,a) reactions on carbon and oxygen nuclei. Using heavy ion kerma spectr < 15 MeV and proton kerma < 10 MeV it was possible to predict Y* values and RBE trends.
Monte Carlo simulation of fast neutron spectra: Mean lineal energy estimation with an effectiveness function and correlation to RBE. (J-P Pignol, J P Slabbert, P Binns)
To get a better understanding of the influence of neutron energy on radiation quality, Monte Carlo simulations were performed to calculate fast neutron spectra at different irradiation positions for different neutron therapy facilities. Using an effectiveness function, the fast neutron spectra could be correlated with the mean lineal energies obtained experimentally by other investigators. The effectiveness function appears to be a suitable parameter to predict variations in RBE at the various neutron therapy facilities.
Treatment of an ovarian cancer cell line using 123I labelled to monoclonal antibodies. (H Neethling, E Truter, J P Slabbert).
Antibodies specific to a DMBA cell line were first raised in culture, then purified from the harvest fluid using a hydroxyapatide column. Iodogen reagent was then used to conjugate 123-Iodine to these antibodies. Specific binding to cells were noted as approximately 8 times more radioactivity could be measured compared to cells exposed to equivalent levels of [123I]NaI. From this it clear that the antibodies will be very suitable for nuclear medicine imaging of ovarian cancers. It is however unclear if sufficient levels of activity can be delivered with this method for therapeutic purposes.
Neutron radiation sensitisation with a halogenated pyrimidine. (KK Nkuna, J P Slabbert, A Lennox, P Joseph, A Hendriks).
Following our earlier observations that many human tumour cell types are resistance to neutrons, the usefulness of sensitisation with a halogenated pyrimidine (BrdU) has been investigated. Neutron dose modifying factors measured in vitro are similar to that seen for gamma rays. This indicates that a halogenated pyrimidine would be a suitable compound for use in neutron therapy of fast growing radioresistant brain lesions. In vivo testing has also been initiated using a radioresistant rhabdomyosarcoma model. Although extensive delays in tumour growth have been observed, radiosensitisation by BrdU was only marginal. Monoclonal antibody labelling of tumour sections show poor drug up-take and in part explain the findings.
Apoptosis in response to neutron and gamma ray damage. (A Rossouw, K. Smit J P Slabbert, K Meehan).
Apoptosis in response to neutrons and gamma rays has been measured in human lymphocytes using caspase 3 and 7 activities. Neutrons constantly result in less apoptotic response when followed using this molecular marker. By contrast, direct microscopic observations reveal clear dose response relationships with a higher apoptotic cell frequency in neutron-irradiated samples.
Single Cell Gel Electrophoreses (SCGE) as a measure of radiosensitivity. (K Smit, J P Slabbert, K Meehan)
Modern methods to quantify cellular damage as a result of radiation injury are also under investigation. Single cells gel electrophoresis of proves to be most useful with an epithelial cell type but DNA damage is similar and even less in human lymphocytes when comets from neutrons are compared to that from gamma irradiated cells.