Cell damage after X-ray irradiation

Jörg Pawelke, Elke Beyreuther, Wolfgang Wagner

In 1895 Wilhelm Conrad Röntgen discovered the X-rays which have been immediately and enthusiastically applied for medical diagnostics. The side effects thereby observed such as skin damage or loss of hair have triggered interest in both the radiobiological properties of X-rays and their use for therapeutic irradiations with a first treatment performed already in 1896. More than 100 years after their discovery X-rays are still the subject of radiobiological research.

In vitro cell experiments allow the study of basic radiobiological effects and mechanisms following an exposure of living cells to ionising radiation. Such experiments revealed the DNA being the main target for the radiation causing biological effects in human cells. Furthermore, these effects are influenced amongst other things by the dose and quality of the radiation. The second fact is expressed by the quantity of the relative biological effectiveness (RBE), the ratio of the absorbed doses of two types of radiation that produce the same specific effect. While .-rays were declared to be the reference radiation, an RBE value of 1 was assigned to photon radiations of all energies. However, in vitro studies have already shown that, especially at low doses, low-energy X-rays possess an increased biological effectiveness (RBE > 1) compared to high energy photons. These differences are of interest in themselves, but must also be taken into account when different photon radiations are used as reference radiation or applied in radiation therapy and diagnostics.

The study of the RBE dependence on photon energy by in vitro cell irradiations is part of the radiobiological research at the Forschungszentrum Dresden-Rossendorf and subject of a collaboration with the Technische Universität Dresden. This interdisciplinary topic is bringing together the expertise from several fields necessary:

(i) operation of an appropriate radiation source, (ii) physical characterisation of the radiation field, (iii) irradiation of probes of living cells including an accurate determination of the dose absorbed by the cells and (iv) suitable methods of determining the radiation-induced biological effects in cells on cellular and molecular level.

Effectiveness of mammography X-rays

The reason for these studies was an insufficient knowledge of the RBE of low energy X-rays (below about 30 keV) which are mandatory for mammography because of the necessary tissue contrast. Before establishing nation wide screening programs the risk-benefit ratio has to be reliably estimated. The RBE determined for various mammalian cell lines range from less than one up to about eight and depends on cell line, biological endpoint and applied reference photon radiation. Furthermore, no experimental studies had been performed on human mammary cells so far, although these are the cells of interest for the induction of breast cancer.

Therefore, two human mammary epithelial cell lines have been chosen to determine the RBE of 10 kV and 25 kV soft X-rays relative to 200 kV X-rays, all generated by conventional X-ray tubes. Photon energies below 10 keV are less relevant to exposure situations in human due to their strong attenuation in tissue, but are of particular interest with regard to biophysical considerations and to show the trend of energy dependence. Various endpoints have been investigated, so the clonogenic cell survival and several types of DNA damage including the repair kinetics of DNA double strand breaks (Fig. 1). The results [1-3] confirm a moderately increased RBE of mammography X-rays in the range of 1 – 2, but not the high values of a recent claim, based on an experiment on cell transformations in a human hybrid cell line and on re-interpretation of various earlier RBE data. Comparable RBE values have been determined for both mammary cell lines, displaying the same photon energy dependence (Fig. 2) and being consistent with experimental data achieved for human lymphocytes as well as with results of several theoretical calculations of RBE.

Fig. 1 Mammary epithelial cell damaged by X-ray irradiation with a dose of 2 Gy: chromosomal damage shown by the induced micronuclei (upper left), chromosomal aberration (tricentric) determined by fluorescent plus Giemsa staining (upper right), chromosome specific aberration (two translocations involving chromosome 1, pink colour) observed with the method of fluorescence in situ hybridisation (bottom left) and DNA double strand breaks indicated by the formation of .-H2AX plus 53BP1 foci (green colour, bottom right).

Fig. 2 Yields of chromosomal exchange aberrations per cell induced by the different X-ray qualities in dependence on dose.

Since any material included in the beam can substantially modify the dose absorbed in the cells at these low photon energy values, the implementation of an accurate dosimetry [4-6] considering the exact irradiation conditions was important for the reliability of the data. Thanks to the determination of the X-ray spectra, the contribution of the different energy ranges to the dose could be calculated and compared. Low-energy components are present in the broad X-ray spectrum (Fig. 3), and the biological effects of the different photon energy ranges are superimposed.

Fig. 3 Energy distribution of an X-ray tube with tungsten anode operated at 25 kV.

Intense, monochromatic channeling X-rays at ELBE

Experiments on an X-ray tube cannot provide entirely photon-energy resolved information on the biological effectiveness. Channeling radiation (CR), which is emitted by relativistic electrons during their passage through a diamond crystal parallel to a crystal plane (Fig. 4), has been proclaimed to be a bright and tunable monochromatic X-ray source already in the 1990s. However, such a nonconventional X-ray source, dedicated for practical application, has worldwide for the first time been realised at the Radiation Source ELBE.

Fig. 4 Scheme of Channeling X-ray generation.

The yield and spectral distribution of planar CR have been measured in dependence on the crystal properties as well as parameters of the ELBE electron beam at low beam current (several nA) [7-10]. The results show that by variation of the electron beam energy the photon energy can be tuned in the range from 10 keV up to about 100 keV and favour thicker diamond crystals if large CR intensities are required. An intense CR source dedicated for radiobiological application was designed and implemented at ELBE [11]. The commissioning has proven its stable operation over hours with an average electron beam current of up to 100 µA, which allows to reach photon rates of quasi-monochromatic CR of the order of 1011 s-1. For this, various technological challenges had to be solved: (i) precise crystal alignment with respect to the beam axis by a newly constructed goniometer, (ii) water-cooling of the crystal while guaranteeing that the electron beam passes the 150 µm thick diamond crystal of 4 x 4 mm2 size, (iii) on-line monitoring of the photon-energy spectra at high beam current by means of a Compton spectrometer and (iv) X-ray monochromatisation applying Bragg diffraction at a highly ordered pyrolytic graphite (HOPG) crystal. Here, monochromatisation means to filter out some part of the spectral distribution at such photon energies, where the CR line superimposes the broad polychromatic bremsstrahlung background naturally associated with CR production (Fig. 5). Cooperation with industrial partners was essential for the success. So the unique HOPG technology of Bourevestnik Inc., St. Petersburg, Russia, allowed to design and build a special toroidal HOPG X-ray reflector which focuses the X-rays to the probe for irradiation of living cells.

Fig. 5 Spectra of quasi-monochromatic channeling radiation with the associated bremsstrahlung (blue line) measured for 14.6 MeV electrons channeled in the (110) plane of a 42 µm thick diamond crystal. The narrow peak (red line) represents monochromatic X-rays after Bragg reflection at the (002) plane of a planar large-area HOPG crystal.

Radiobiological experiments are planned at the ELBE X-ray beam for 2007.

[1] “RBE of 25 kV X-rays for the survival and induction of micronuclei in the human mammary epithelial cell line MCF-12A”, A. Lehnert, E. Leßmann, J. Pawelke, W. Dörr 1, 2, Radiat. Environ. Biophys. 45 (2006) 253

[2] “RBE of 10 kV X-rays determined for the human mammary epithelial cell line MCF-12A”, A. Lehnert, E. Leßmann, J. Pawelke, W. Dörr 1, 2, Radiat. Res. (2007) submitted (accepted ?)

[3] “RBE of 25 kV and 10 kV X-rays for the induction of chromosomal aberrations in two human mammary epithelial cell lines”, E. Beyreuther, W. Dörr 1, 2, A. Lehnert, E. Leßmann, J. Pawelke, Carcinogenesis, (2007) submitted

[4] „AMOS - An effective tool for adjoint Monte Carlo photon transport”, D. Gabler 3, J. Henniger 3, U. Reichelt, Nucl. Instrum. Meth. B 251 (2006) 326

[5] „Application of advanced Monte Carlo methods in numerical dosimetry”, U. Reichelt, J. Henniger 3, C.Lange 3, Radiat. Prot. Dosim. 119 (2006) 479

[6] “Investigation of a TSEE dosimetry system for determination of dose in a cell monolayer”, A. Lehnert, E. Beyreuther, E. Leßmann, J. Pawelke, Radiat. Meas. (2007) in press

[7] “Channeling X-rays at the ELBE radiation source”, W. Wagner, B. Azadegan, A. Panteleeva, J. Pawelke, W. Enghardt 2, Proc. SPIE 5974 (2006) 0B-1

[8] “Electron beam monitoring for channeling radiation measurements”, W. Neubert, B. Azadegan, W. Enghardt 2, K. Heidel, J. Pawelke, W. Wagner, Nucl. Instrum. Meth. B 245 (2007) 319

[9] “Dependence of the linewidth of planar electron channeling radiation on the thickness of the diamondcrystal”, B. Azadegan, W. Wagner, J. Pawelke, Phys. Rev. B 74 (2006) 045209-1

[10] “Planar channeling radiation from electrons in quartz”, W. Wagner, B. Azadegan, L.Sh. Grigoryan 4, J.Pawelke, Europhys. Lett. (2007) in press

[11] “An intense channeling radiation source”, W. Wagner, J. Pawelke, B. Azadegan, M. Sobiella, J. Steiner, K. Zeil, Nucl. Instrum. Meth. B (2007) submitted

Project partners:

1 Klinik für Strahlentherapie und Radioonkologie, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Germany

2 Zentrum für Strahlenforschung in der Onkologie (OncoRay), Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Germany

3 Institut für Kern- und Teilchenphysik, Technische Universität Dresden, Germany

4 Institute of Applied Problems of Physics, National Academy of Sciences, Yerevan, Armenia