Measurement of the effect of ionising radiation on cells
Established Radiobiological Methods
|Biological edpoints||Specificity of the test|
|Measurement of gamma-H2AX foci||DNA-Repair|
|Micronucleus test||Chromosomal damage|
|Chromosomal aberrations||Chromosomal damage|
|Fluorescence in situ Hybridisation||Damage of certain chromosomes|
|Cell survival||Clonogenic cell death|
The radiation energy is transferred by energy- and material-dependent interactions. For photons, only the processes of absorption and scattering play a role in the low-energy range. Only the absorbed fraction of the radiation in a biological object can produce an effect. The most important changes that can be caused by ionizing radiation in a cell, are the changes in the genetic material contained in the deoxyribonucleic acid (DNA). As a consequence of the primary physical processes, a large number of chemical changes appear in the DNA. The most important breaks can occur in the sugar phosphate backbone, particularly if they arise in both strands in a close proximity to each other ("double-strand breaks", dsb). They are reparable only to a certain extent and can lead to cell death, moreover, when not correctly repaired, are the starting point for chromosomal aberrations, mutations and neoplastic transformations.
One of the first measurable reactions of the mammalian cell to dsb induction is the phosphorylation of the Histons H2AX in the vicinity of the break to the so-called gamma-H2AX (Rogakou et al. (1999) J Cell Biol 146:905-16). Using specific antibodies, gamma-H2AX foci can be demonstrated already few minutes after irradiation. It has been shown that the number of gamma-H2AX foci very well correlates with the number of dsb shortly after irradiation (Rothkamm and Löbrich (2003) Proc Natl Acad Sci USA 100:5057-62). The following reduction of the foci number is connected with the repair processes.
Human mammary epithelial cells 184A1, irradiated with 200 kV X-rays, 2 Gy
The ionising radiation leads to the formation of both numerical (deviations in the number of chromosomes) and structural chromosomal aberrations (changes of the chromosome structure). A simple method to study chromosomal damage is the micronucleus test. In order to distinguish the cells to make clear that have passed exactly one division after irradiation, cytochalasin B is added to the growth medium in order to block the cytoplasm division, however not the division of the nucleus. Thus so-called binucleated cells appear. The chromosome fragments or whole chromosomes, eliminated during the cell division, are then visible in the cytoplasm as micronuclei (MN).
Mouse fibroblast NIH/3T3, irradiated with 4 Gy of 200 kV X-rays. The arrow points to a micronucleus.
In order to study chromosomal aberrations, the cells are observed before the completion of the cell division, in the metaphase. The simplest chromosomal change is the so-called break, which develops into an acentric fragment. Changes, in which two breaks are involved, can lead to unstable and stable aberrations, referring to whether the cell survives or dies due to this chromosome aberration. The so-called dicentric chromosomes and rings belong to the first group. Since the spindle apparatus can attach at two sites of a dicentric chromosome, in half of all divisions this leads to the death of the cell. These aberrations are easy to recognise by means of simple staining of all chromosomes (Giemsa staining).
Human mammary epithelial cell MCF-12A, irradiated with 10 kV X-rays, 5 Gy. An acentric fragment and a centric ring are shown by arrows.
Human mammary epithelial cell MCF-12A, irradiated with 10 kV X-rays, 5 Gy. Four acentric fragments and four a dicentric chromosomes are shown by arrows.
On the other hand, the stable chromosome aberrations are not connected with loss, but only with a relocation of genetic material. To this group belong inversions and translocations. Although they do not lead to cell death, serious genetic effects, which can lead to neoplastic transformation, can occur. Since the Giemsa staining results in homogeneous staining of chromosomes, these aberrations can not be recognized. In this case, the "FISH" (fluorescence in situ hybridization) staining is applied. By specific staining of individual chromosomes, the translocations are easily recognized.
A metaphase of the human mammary epithelial cell line 184A1, irradiated with 10 kV X-rays, 3 Gy. Chromosome 1 (red), 8 (green) and 17 (orange) are stained. Chromosome 1 is involved in a translocation.
The radiotherapy of tumours is based on the preventing the supply of cells, which normally takes place via division and differentiation. These processes can be examined also in cell cultures, since the ability of the cells to form colonies is reduced by irradiation in a dose-dependent way.
Colonies of the mouse fibroblast cell line NIH/3T3, irradiated with 6 Gy of 200 kV X-rays (left) or not irradiated (right)