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Bioradiation

الكلية كلية العلوم     القسم قسم علوم الحياة     المرحلة 3
أستاذ المادة اياد محمد جبر المعموري       10/26/2011 11:28:18 PM
-lect3
Bioradiation

Quantifying Radiation Effects
Comparison of the effectiveness of different radiation types requires an accurate
quantification of radiation effects. This section describes the basic experimental
procedures which are used in classical radiobiological studies, covering different
biological levels including DNA strand breaks, chromosome aberrations, and cell
death as defined by the loss of proliferative capacity.

Strand Breaks
Direct investigation of DNA strand break induction is often performed using particularly simple, small DNA molecules from viruses or plasmids. These systems have the advantage that induction of SSB or DSB leads to characteristic topological changes of the molecule, which allow one to identify unambiguously the fraction of molecules containing no damage at all, a single strand break, or a double strand


Top: Induction of strand breaks in viral and plasmid DNA. The undamaged (native) form has a supercoiled structure. Induction of a single strand break leads to an open ring form, whereas by induction of a double strand break the molecule is transformed into a linearized form. The linearized form can also result from induction of two single strand breaks in close vicinity Bottom: The three conformations are characterized by different migration velocities in the gel.






Studying strand breaks in mammalian cells is more demanding. Whereas the
size of viral or plasmid DNA is in the order of some thousand basepairs (bp),
mammalian cells typically contain in the order of 3 109 bp. Inducing approximately 350 DSB by irradiation with 10 Gy – which is in the order of typical doses \applied in cell experiments – roughly corresponds to 1 DSB/107 bp. This is much lower than the value of approximately 1 DSB/104 bp which is the order of magnitude for investigations using plasmid DNA. In addition, induction of DSB in mammalian DNA does not lead to defined conformational changes of the DNA molecule as in plasmid DNA. Therefore, the number of strand breaks has to be es- timated from the production of DNA fragments. According to the random distribution of energy deposition events (see below) a random distribution of breaks within the genome can be expected in a first approximation, and fragment sizes will cover a correspondingly broad spectrum. Small fragments (<6–9 Megabasepairs) can be separated from large fragments and undamaged DNA by gel electrophoresis. According to the broad spectrum of fragment sizes, fragmented DNA appears as a broad smear

From the fraction of small sized DNA, the number of DSB can then be estimated using appropriate calibration procedures.

Gelelectrophoretic detection of double strand breaks in cellular DNA. Intact DNA as well as large fragments are retained in the plugs, whereas smaller fragments migrate in t he gel . The total amount of DNA detected in the broad smear below the plug is a measure of the number of induced DSB; the width of the distribution depends on the fragment length distribution. Bottom: Rejoining of DSB can be detected by measuring the kinetics of the fraction of small fragments as a function of time after irradiation. During the incubation interval, strand breaks are rejoined and thus larger fragments are reconstituted, so that the fraction of residual DSB decreases with time.
Gel electrophoretic methods are also suitable to study the processing of DNA
strand breaks such as, e.g., rejoining of the open ends by measuring the fraction of DNA fragments as a function of time after irradiation. During incubation,
strand breaks can be repaired or rejoined, so that larger size DNA fragments are
reconstituted from smaller fragments. Repair and rejoining have to be distinguished here, since with the methods described here the restitution of DNA length can only be detected within certain limits, but the loss of very small fragments of DNA during the rejoining process cannot be detected. Figure 4b gives a typical example of such a rejoining curve after photon irradiation. Decay of the fraction of damaged DNA obeys an exponential law with a fast and a slow component. Rejoining of strand bre aks is not always complete and, depending on radiation type and cell line, a certain fraction of residual damage can be observed even after long intervals of incubation. This residual damage is frequently used as indicator for the lethality of a given radiation type









Chromosome Aberrations

From the rejoining of DNA DSB no conclusions about the fidelity of the rejoining
process can be drawn in general. A more detailed study of misrejoining and misrepair processes can be performed based on the analysis of chromosome aberrations. The advantages of using chromosome aberrations as indicators for radiation effects are that doses as low as 0.05 Gy can be detected, and that aberrations can be directly observed in the microscope Since the duration of mitosis, in which the DNA becomes extremely condensed and microscopically visible as chromosomes, is very short (20–30 min) compared to the complete cell cycle duration (typically 12–24 h), only a small fraction of cells can be found in mitosis at a given time interval. In order to increase the yield of mitotic cells, cell populations are treated using blocking agents like, e.g., colcemide, up to mitosis, so that with increasing time (typically 2 h) mitotic cells accumulate. which prevent cells from completing mitosis, but do not affect the proliferation.

An example of a radiation induced chromosome alteration is shown inbelow figure these ‘dicentric’ chromosomes are often used as marker for lethality. schematically explains how these aberrations can occur by misrejoining of two
DSB produced in two adjacent chromosomes. The lethality of dicentric chromosomes is attributed to the fact that during cell division the correct segregation of these chromosomes and the accompanying acentric fragment is not guaranteed, so that genetic information is not symmetrically distributed between the daughter cells.
Although direct visibility of chromosome aberrations is a significant advantage,
it has to be taken into account that not all types of aberrations become visible. For example, if approximately equally sized pieces of DNA are exchanged between chromosomes (which can happen if two DSB are produced in each of the two chromosomes, respectively), this exchange could not be detected by means of standard staining techniques However, using more advanced
techniques like fluorescence in situ hybridization (FISH), it is possible to stain selectively the DNA of one particular chromosome with a distinct fluorescence color or even to stain each of the chromosomes with a different color, so that any complex type of exchange of DNA chromosome pieces will become visible




Radiation induced chromosome aberration. The arrow indicates a dicentric chromosome. This aberration type is often used as marker for lethal lesions. The
schematic drawing below illustrates the mechanism leading to the formation of dicentric chromosomes by misrejoining of chromosome fragments resulting from two DSBs in two different chromosomes in close vicinity.

Cell Survival
Investigation of radiation induced cell death, defined as mitotic death in the sense of a complete loss of the proliferation capacity, is one of the most commonly used methods to study radiation effects on cells. As mentioned earlier, many cell types are characterized by regular cell division in 12–24 h intervals. Thus, according to the exponential growth, a single cell can produce thousands of daughter cells within a few days. If the cells are originally seeded in culture flasks at the appropriate low density, the daughter cells of each individual cell appear as clusters or ‘colonies’. A cell is classified as ‘survivor’ if it is able to produce at least 50 daughter cells within a time interval of approximately 10–14 normal division cycles, i.e., 5–14 days; if less than 50 daughter cells are produced the cell is classified as dead or ‘inactivated’ [35]. The threshold of 50 cells is an empirically determined value and somewhat arbitrary; actually there is no clear-cut value defined because there is a smooth transition between cells producing no daughter cells at all and cells producing the maximum possible value of 210–214 daughter cells.
Most experiments to study survival probabilities are based on a so-called dilution
assay, which briefly consists of the following steps (see below figure)
*After irradiation, a cell suspension is produced by removing the cells grown on
the bottom of the culture vessel by controlled enzymatic digestion. The cell
number in the suspension is counted.
* From the dose delivered, the expected fraction of surviving cells is estimated. The cell suspension is then diluted and aliquots are reseeded to new culture vessels at a density, that approximately 100 surviving cells are expected per culture vessel.
* Cells are incubated for 5–14 days typically, corresponding to 10–14 cycle times.
* The number of colonies with more than 50 cells is determined; the fraction of
surviving cells is then calculated by normalization to the number of cells originally seeded in the flask.




Dilution assay for measuring cell survival after irradiation (for details see text). The insert shows typical dose response curves observed after irradiation with photon radiation of normal, repair-proficient cells (full line) and repair-deficient, sensitive cells (dashed line)




The shoulder shape indicates that the efficiency of radiation increases with
dose, which can be attributed to the more complex and thus less reparable damage induced at higher doses. This higher complexity can be explained by the interaction of damage produced in close vicinity: the probability to induce multiple ‘sublethal’ damage in close vicinity increases with increasing dose. This view is further supported by the results obtained with cell types containing genetic deficiencies, e.g., in DNA double-strand break repair. The loss of repair capacity is reflected in a higher overall sensitivity of the cells on the one hand, but also in a different shape of the survival dose response curve on the other hand: the repair deficient cells show a more or less straight line dose effect response .The reduction of the shoulder can be attributed to the loss of repair capacity; due to this loss, even non- complex damage cannot be repaired, so that already low doses exhibit a comparably high efficiency of cell killing.

Modification of Radiosensitivity

Several environmental factors such as, e.g., temperature, pH, or oxygen supply
have significant impact on radiosensitivity. Figure below illustrates the effect of oxygen by comparing survival curves obtained under normal oxygen supply and under hypoxic conditions,









































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