Induction of DNA strand breaks by heavy ions

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B 107 ( 1996)

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Induction of DNA strand breaks by heavy ions G. Taucher-Scholz *, J. Heilmann, G. Kraft Gesellschaft ftir

SchwerionenforschunK,

Postfach 11032,

D-64220

Damtadt,

Germany

Abstract The cellular response to ionizing radiation is mainly determined by damage to DNA. DNA double-strand breaks (DSBs) constitute critical lesions, which if not correctly repaired can lead to mutation, transformation or cell death. Aimed at understanding the relationship between the induction of these lesions by radiations of differing qualities and biological effects, DSB yields have been evaluated in mammalian cells following heavy ion irradiation. The initial production of DSBs was found not to vary significantly with increasing radiation quality over a range where biological effectiveness varies widely. This fact can be explained assuming that lesions of different complexity, all monitored as DSBs by current assays, are induced by radiation of differing LET. Indirect evidence supporting this notion arises from rejoining studies, showing slower kinetics and a reduced extent of DSBs rejoined with increasing LET Therefore, inadequate processing of a clustered type of DSBs, arising from the high local ionization density within the particle track, may contribute to cell lethality after particle irradiation. Studies on DNA strand break formation using small DNA molecules in radioprotective solution also support the concept of clustered breaks.

1. Introduction Understanding the mechanisms involved in radiobiological effects of heavy ions has become increasingly important in view of the application of accelerated particles in radiation therapy of turnours. The requirement of radiation risk estimates as well as radiation protection in space are also subjects of major concern. Being the carrier of the whole genetic information which is passed on from one cell generation to the next, it is generally accepted that nuclear DNA constitutes the principle target for ionizing radiation damage to cells (for review see [ 1] ). It is a large and unique molecule, and a defect or loss of essential genetic information cannot be compensated. Thus, a complex repair machinery operates in the cell in order to maintain the integrity of DNA [ 21. However, the rupture of the DNA backbone at one strand or, most critically, at both strands of the double-helix constitutes a very severe, in some cases lethal event. The relationship between the initial radiation induced lesions and the various cellular endpoints like cell death, chromosome aberrations, mutations or transformation, is still not fully understood. The effectiveness for cell killing has been shown to vary significantly with increasing linear energy transfer (LET, numerically equivalent to energy loss dE/dx). For LET values around 100 keV/pm the relative biological efficiency (RBE) for cell inactivation is up to 4-10 times higher than for sparsely ionizing radiation, although it should be kept in * Corresponding

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mind that biological effects depend not only on the energy deposited but also on the distribution of ionization events as determined by the particle track structure (for reviews see Refs. [ 3-51) . Therefore, the examination of DNA doublestrand breaks (DSBs) as a function of LET is an important task in radiation biology. The use of particles provided by accelerators has allowed to investigate the influence of radiation quality on the induction of DNA damage, in particular strand breaks, but also chromosome aberrations and mutations, by varying both energy and atomic number of the projectile to cover a wide LET range up to 14 000 keV/,um [6]. The targets used to study the effects of heavy ions on DNA range from simple polynucleotides and small plasmid molecules up to genomic DNA of bacteria or even eukaryotes. In the present contribution we will first focus on the determination of DNA strand breaks in model systems of SV40 viral DNA in solution, and then discuss the induction of DNA double-strand breaks in mammalian cells. While the former system allows accurate measurements of both single (SSB) and double (DSB) strand break yields, the cellular environment and the damage processing component are advantages of the in viva system.

2. Measurement

of DNA strand breaks in vitro

The irradiation and analysis of small, defined DNA molecules in solution is well suited for studies of the action of heavy charged particles on a molecular level. In partic-

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LET1oIkeV/p;l Fig. I. Cross sections for the induction of SV40 single strand breaks (SSB) and double strand breaks (DSB) versus LET. Ion species used for irradiation are indicated at the bottom of the panels. SV40 DNA was exposed either in 10 mM Tris-buffer (TE) or upon addition of 100 mM mercaptcethanol (EtSH) UCradical scavenger [ 29 1. The dotted lines represent the RBE = I curve (see text).

ular, the influence of an inhomogeneous dose distribution within particle tracks on the production of SSBs and DSBs in the same molecule can easily be monitored. The DNA target consists of a circular plasmid or viral DNA, which natively exists in an overtwisted, very compact supercoiled conformation. One SSB allows relaxation and one DSB produces a linearized molecule, and the three DNA conformations can be distinguished on the basis of their different electrophoretic mobility on agarose gels [ 7). From the rel-

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ative amounts of each form after irradiation, dose or fluence effect curves can be established and cross sections for SSB or DSB induction can be calculated. With this kind of experimental system the effect of particle irradiation on SSB and DSB yields has been studied using @Xl74 DNA [ 81 or plasmid DNA [ 91. The most extensive compilation of data for heavy ions exists for SV40 DNA irradiated in a very dilute solution [ lo] or upon addition of radical scavengers to mimic the cellular environment [ 1I]. Generally, action cross sections increase with LET. However, the pattern of LET dependence obtained [lo] shows a less than proportional increase of cross section with LET up to 500 keV/pm for SSBs and there is a similar tendency for DSBs (in DNA under non-protective conditions). In the LET region corresponding to heavier ions, for SSBs and DSBs the a-LET relationship splits into individual curves for each atomic number, showing a plateau and subsequent decrease of a-values with increasing LET. An experimentally not apparent local overproduction of strand breaks and/or recombination processes within regions of high ionization density may lead to this decrease in strand break effectiveness for the low energy high LET particles, giving rise to characteristic hooks [5,7]. This feature also occurs for other endpoints and has been extensively discussed [4,5,12]. In order to study the influence of the chemical environment on strand break induction, SSB and DSB production cross sections for SV40 in dilute solution and in the presence of scavengers are presented in Fig. 1. The main feature inferred from this dataset, in addition to the points discussed above, is the overproportional increase of DSB cross section with LET observed between 200 and 2000 keV/pm in the protective solution only, although strand breaks yields are generally reduced upon addition of radical scavengers [ 131. Around 500 keV/pm, the efficiency for DSB induction per unit dose shows a maximum and is significantly higher than for X-rays, in agreement with previous data [ 81. This may be explained by the correlated induction of two SSBs in close proximity on opposite strands of the DNA, leading to a DSB by denaturation of the few base pairs in between. In regions of the particle track with high local ionization density such a process is likely to occur.

3. Measurement of double-strand mammalian cells

breaks in

The genomic DNA of cells, orders of magnitude larger than the simple molecules discussed above, resides in the nucleus in a highly condensed form achieved by folding and packaging with proteins [ 141. In such a complex system, the direct measurement of single SSBs or DSBs is not possible. However, several techniques have been developed from which DNA DSB yields can be derived [ I]. Recently, the electrophoretic elution of radiation induced DNA fragments out of agarose plugs has emerged as a valuable tool

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Fig. 2. Pattern of gelelectrophoreticelation of DNA fragments following CHO cells to increasing fluences of 265 MeV/u C-ions of LET 14 keV/pm from lanes 1 to 9. Lane IO is the onirradiated control. The correspondingfluence-effect curve is shown below (triangles). Effect curves for irradiation with 5.4 MeVlu (228 keV/Fm) and 18 MeV/u ( 103 keV/pm) C-ions are also included in the graph, to show the great effectivenessin DSB production by the low energetic particles.

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for evaluating DSBs [ 151. Several variations of pulsed field gel electrophoresis (PFGE) have been applied [ 16-191, but constant tield gel electrophoresis (CFGE) is suitable as well [20,21]. To exemplify the CFGE elution, a typical gel obtained after irradiation of Chinese hamster ovary (CHO) cells with C ions is presented in Fig. 2. From the fraction of high molecular weight DNA retained in the well, fluence effect curves are established as shown in the figure. The shape of the curve has a methodological basis [ 22,231 and corresponds to a linear induction of DSBs. The fraction of DNA retained can be converted into DSB induction by “folding” with the X-ray dose effect curve, assuming an absolute DSB yield of 22 DSB/Gy per cell as derived previously for the CHO-K 1 cells [ 241. A fraction of 0.7 of DNA retained was taken as the reference for calculation of DSB production cross sections (c~nsa), arbitrarily normalized to a DNA size of 10’ g/mol for the purpose of comparison. The resulting cr-values for DSB induction after irradiation with C ions and

other particles [ 24,251 are depicted in Fig. 3 and discussed below. The effect of heavy ions on DSB induction has also been studied by Kampf (261 in V79 hamster cells for ions up to oxygen, and for heavier ions by Aufderheide et al. [ 271 and Heilmann et al. [ 281. PFGE was applied by Weber and Flentje [ 251 to measure DSBs for neutron and higher LET radiation. Data covering a wide LET range were obtained for CHO cells using CFGE elution [ 291. The intermediate LET range has been studied by irradiation of these cells with high and low energetic C ions [ 231, Human fibroblast cells were exposed to high energetic C and Fe ions and PFGE as well as a novel gene probing method were applied for DSB analysis [ 30,3 11. The influence of radiation quality in the lower LET regime is covered by several studies on DSB induction in mammalian cells following irradiation with protons or (Yparticles [ 32-361. The general systematics inferred from the whole body of information available on high LET DSB induction is in good agreement with the exemplified dataset presented before (Fig. 3). Indeed, Rosa increase with LET, showing up for the high efficiency per particle in producing DSBs for ions like Ar or Xe. In the slowing down region of each particle the dependence on particle energy rather than LET is suggested, as known for other endpoints [ 51, although data are not sufficient to be conclusive. A similar behaviour has also been shown for DSB-induction in yeast cells at high LET [37]. In the LET region around 100 keV/pm however, in contrast to yeast studies [38], no overproportional increase of DSB production cross sections with LET is observed for the CHO-Kl data presented here (Fig. 3). For clarity, the line corresponding to a constant increase of DSB yield (slope= 1). equivalent to a relative biological efficiency (RBE) of unity, is included in the graph. As observed, action cross sections for DSB production are not sig-

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LET [keV/pml Fig. 4. Relative lethality calculated for double-strand breaks induced by C-ions of increasing LET using X-rays as reference. An increase of lethal lesionc per DSB with a maximum around 200 keV/.um is observed. nificantly above the RBE = I curve in any case, and even values below RBE of unity are obtained at LET> 100 keV/pm, in agreement with all of the more recently published results. As the efficiency for cell killing significantly increases with LET being greatest at LET values around 100-200 keV/,um [ 61 but yields of DSBs (per unit dose) remain similar or even decrease, the probability per induced DSB to lead to a lethal lesion is enhanced. From the comparison of cross sections for DSB induction with the corresponding inactivation cross section (for the same radiation type), the relative killing probability per DSB and cell can be derived. This is shown for C ion irradiation in Fig. 4. As already pointed out by similar studies with Lu-particles [ 331 and heavy ions [ 251, lethality of DSBs produced at higher LETS is dramatically increased.

4. Lesion complexity Despite the large body of evidence indicating that DSBs are the critical damage for cellular endpoints, the results obtained fail to correlate initial DSB yields and cell death in the region where cell inactivation is in its maximum. Therefore, differences in the quality of lesions with LET have been postulated as the basis for biological radiation effects [ 39,401. Thus, an increase in the complexity of DSBs could be responsible for the enhanced cell killing efficiency observed at higher LETS [ 4 I 1. In fact, calculations simulating individual ionization events and their spatial correlation at nm dimensions (corresponding to the size of the DNA helix) have revealed that local clusters of ionizations are conceivable within tracks [42] and that the degree of complexity of lesions is likely to increase with LET [ 12,391, giving rise to locally multiply damaged sites [43]. This kind of complex lesion would not be distinguished from a simple DSB by the current assays, but the increase in DSB lethality with LET

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Fig. 5. Rejoining of double-strand breaks measured after exposure of CHO cells to C-ions of 261 MeV/u (LET 14 keV/pm) and 5.4 MeV/u (LET 228 keV/pm). The relative fraction of DNA damage remaining is plotted as a function of postirradiationincubation time under culture conditions.

(Fig. 4) strongly suggests differences between the lesions detected at the various LETS. Besides modelling calculations estimating the clustering of ionizations and multiple damage on DNA [ 44-461, indirect experimental evidence for enhanced high LET lesion complexity has arisen from the reduced capacity of cells to deal with the damage induced. This has been mainly monitored by rejoining studies. While DSBs induced by sparsely ionizing radiation can be rejoined quite efficiently, the repairability of heavy ion induced lesions is significantly reduced [ 2,28,25,29]. As shown in Fig. 5, following irradiation with high energetic C particles (LET 14 keVlpm) and postirradiation incubation, both a fast and a slow kinetic rejoining component are observed, similar to X-irradiation. But for the low energetic C ions (LET 228 keV/,um) only slow rejoining takes place and the fraction of residual damage after a 3 h incubation period is about 3 times higher than for the lower LET ions. In a further investigation [ 30,3 I 1, the rejoining kinetics (which usually does not assess accuracy of repair in any way) is compared to the kinetics of restitution of individual DNA fragments after irradiation with high energetic Ne and Fe ions. In this study, fidelity of repair was reduced at higher LET. Summarizing, increasing evidence is pointing out the unique feature of high LET DNA lesions. Clustering of DNA damage as a consequence of multiple ionization events is suggested to produce complex, less repairable lesions leading to an enhanced biological efficiency of heavy ion cellular effects. However, the molecular nature of these lesions remains to be understood.

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