Ion-beam irradiation provides a promising treatment for some types of cancer. This promise is due mainly to the selective deposition of energy into a relatively small volume (the Bragg peak), thus reducing damage to healthy tissue. Recent observations that electrons with energies below the ionization potential of DNA can cause covalent damage to the bases and backbone have led to investigations into the ability of low-energy (<1 keV·Da−1) ion beams to damage double-stranded DNA. It has been clearly demonstrated that these low-energy ions induce a mixture of single- and double-strand breaks to dried DNA in vacuo. These effects depend upon the number of ions incident upon the DNA, the kinetic energy of the ions and on their charge state. This DNA damage may be important, as all radiotherapies will result in the production of low-energy secondary ions as radiation passes through tissues. Currently, their effects are neglected in treatment planning, and thus more work is required to quantify and understand DNA damage by low-energy ions.
Radiation can be both a cause and treatment for cancer. In either case, it is well established that irreparable damage to cellular DNA is a major factor in the mechanism of its action. In radiotherapy, high-energy radiation is used to induce multiple DSBs (double-strand breaks) in the DNA of tumour cells which leads to cell death . This therapy has been in use for several decades, but has well-documented side effects. If the tumour is buried within the body and irradiation must take place from outside, then the tissue between the source of radiation and the tumour will also be damaged. Although this can be minimized by irradiating from multiple different external sites, it still causes major pain and discomfort to patients. In addition, there is the risk of permanent damage to tissues and organs and even the possibility that the radiation used to cure the cancer may induce secondary tumours.
The promise of ion-beam therapies for cancer
One promising alternative approach is the use of ion-beam radiation in radiotherapy (see, for example, [2–7]). Clinical proton-beam facilities have been in operation for some years worldwide. More recently, heavier ions (e.g. carbon) have been exploited, and facilities have been built in Japan, Germany and Italy. The results from ion-beam therapy are impressive. Clear-up rates are comparable with, or exceed, those achieved with conventional radiotherapy, and the side effects are much lower (reviewed in ). This success is most likely to be due to the physics of energy deposition by different types of radiation. Photon beams (i.e. X-rays and γ-rays) and very light particles (e.g. electrons) deposit their energy in a linear fashion as they pass through tissue. As a consequence, the tissue between the tumour and the surface must be exposed to high doses in order that sufficient energy is deposited in the tumour. In contrast, ion beams deposit very little energy as they pass through tissue until they reach a certain distance. At this point, the so-called Bragg peak, the majority of the energy is deposited and this can be exploited in radiotherapy to ‘focus’ the destructive power of the radiation into the tumour (reviewed in ).
Ions travelling through a condensed medium occasionally collide with particles in the medium giving rise to an ion track. In biomolecular systems, there are two types of collision event: electronic excitation, including ionization and electronic capture by the ion, and direct molecular fragmentation due to the impact. As a result of collision events in an ion track in biological matter, several very reactive chemical species are produced, namely electrons and O•, O•−, H•, OH• and HO2• radicals, along with protons and hydroxide ions . The ions themselves also survive, although with a reduced energy. These species can interact with DNA in many ways. Ions can collide directly with DNA and produce fragmentation in the form of strand breaks , while secondary ions travelling at relatively low velocities can capture electrons from the DNA, thus leaving ‘holes’ or ‘polarons’ that can induce breaks due to changes in the potential energy surface .
DNA damage by low-energy particles
It has been shown recently that photons and electrons with energies below the ionization potentials of DNA can cause covalent damage to double-stranded DNA and its components [13–16]. It is now well established that, in the interaction of low-energy ions with simple molecules in the gas phase, dissociative electron-capture processes become increasingly dominant as the ion energy is decreased below 1 keV·Da−1 [17,18]. (In the physical sciences, energies are often quoted in electron volts, eV, where 1 eV is the energy gained by an isolated electron moving through a potential difference of 1 V. In terms of units more commonly used by biochemists, it is approximately equivalent to 96 kJ·mol−1.) This has important implications for radiotherapy, as energies in this range have previously been disregarded in damage models. However, it is now accepted that low-energy particles are one of the causes of covalent modification to biological molecules on exposure to radiation (reviewed in ).
Low-energy ions cause covalent damage to DNA
Experimental work by ourselves and others has clearly demonstrated that low-energy ions can cause strand breakage in dried DNA in vacuo. Lacombe et al.  showed that low-energy (<5 keV) Ar+ ions could cause both SSBs (single-strand breaks) and DSBs to the plasmid pUC18 dried on to a gold surface. The predominant form of damage was SSBs; however, the fraction of DSBs increased with the ion kinetic energies, up to a limit of approx. 4 keV .
Plasmid DNA is an ideal system in which to identify and quantify types of DNA damage. As generally prepared by either CsCl gradients or commercially available kits, plasmid DNA is isolated largely in a supercoiled form. A SSB converts the supercoiled form into the so-called open circle form, whereas a DSB converts supercoiled into linear DNA (Figure 1). The supercoiled, open circle and linear forms have different mobilities on gel electrophoresis in the presence of an intercalating agent such as ethidium bromide (despite having very similar molecular masses). Consequently, they can be readily separated and distinguished [21,22]. Furthermore, with suitable gel-imaging equipment, it is possible to quantify the ratio of each form present and thus estimate the relative contributions of SSBs and DSBs. In doing so, care should be taken to correct for the differential binding of the various forms to ethidium bromide [22,23]. In addition to damage pathways in which each plasmid molecule suffers one SSB or one DSB, it is possible for MDSBs (multiple double-strand breaks) to occur. Since these are likely to occur essentially randomly throughout the plasmid sequence, they will result in fragments with a wide range of different sizes. They are thus unlikely to be observed on gel electrophoresis (or, at best, result in a smear occurring at greater mobilities than the supercoiled form). However, the occurrence of MDSBs can be inferred from the loss of detected material compared with controls. These plasmid-based assays are subject to considerable experimental variation [20,24]; however, this can be minimized and controlled by collecting numerous replicates for each data point.
Schematic diagram showing the principle of the plasmid-based assay for detecting DNA damage
Both singly and doubly charged carbon (C+ and C2+) ions cause strand breakage in dried pBR322 in vacuo at low energies (1–6 keV) [24–26]. The magnitude of this effect depends upon the number of ions incident on the plasmid, the kinetic energy of these ions and their charge state. MDSBs represent the main type of damage observed in these experiments (Figure 2) . As expected, increasing the numbers of ions incident on the plasmid molecules resulted in more damage, with this effect saturating at approx. 1000 ions per plasmid [24,25]. However, increasing the kinetic energy of the ions did not result in a greatly increased proportion of the plasmids suffering damage. For the same number of incident ions, the fraction of plasmids in the undamaged supercoiled state was essentially unchanged in the range 1–6 keV; however, the fraction of molecules undergoing MDSBs increased relative to those suffering SSBs or DSBs . This suggests that, at higher kinetic energies, the ions have the ability to cause more severe damage when they collide with DNA. Comparison of the effects of C+ and C2+ ions of identical kinetic energy (2 keV) demonstrated that the more highly charged C2+ ions (which possess greater potential energy as a consequence of their additional charge) cause more damage. Furthermore, a greater fraction of this damage results in MDSBs (31% with C2+ at this energy, compared with 5% with C+) [24,26]. This result suggests that, in the case of both singly and doubly charged ions, the potential energy contributes to strand breakage. Chemically, this makes sense as the neutralization of the ions' charge by abstraction of electrons from the DNA is likely to be a highly energetically favoured process.
DNA damage by low-energy ions: effects of ion numbers and kinetic energy
Although this work clearly establishes that ions with low-keV kinetic energies can cause covalent damage to DNA, many questions remain. To date, only a limited range of ions have been studied. It would be interesting to know whether other ions cause damage with similar dose- and kinetic-energy-dependencies. In particular, studying species which are more energetically stable in their charged (compared with uncharged) state would enable investigation of the hypothesis that the potential energy of the ions is a major contributor to stand-breakage reactions. The precise mechanisms of strand breakage remain elusive. It may be possible to address these by irradiating short oligonucleotides of defined sequence and analysing the fragments produced by MS. To this end, novel MS systems in which gas-phase targets can be irradiated and then analysed within the same instrument will assist in this line of experimentation [27,28].
Since ions with these low energies have very low-penetration distances in air, the experiments described must, of necessity, take place in vacuum. Although this yields fundamental information about the interactions between ions and biomolecules, it will also be necessary to study more physiologically realistic situations. in vivo, DNA is hydrated, and the water surrounding DNA is an important mediator of damage. Ionizing radiation can interact with water molecules, cleaving them to produce ions and radicals which go on to damage DNA . Thus the design of experimental systems which incorporate water is a priority.
Ultimately, of course, we wish to know whether ion beams in the low-keV energy range can cause irreparable damage to living cells. There is some recent evidence to suggest that this is so. Bystander effects have been observed in Arabidopsis seeds irradiated with 30 keV Ar+ ions . These effects could be reduced by the inclusion of radical scavengers such as DMSO, suggesting that reactive oxygen species may be involved in the mechanism. Protons at 35 MeV have been shown to kill mammalian cells through a mechanism which involves reactive oxygen species and apoptotic pathways . Designing experimental systems which can be used to investigate the effects of lower-energy ions will be challenging. Nevertheless, to gain a complete understanding of how they damage DNA, it will be necessary to integrate fundamental biophysical studies in the solid- and gas-phases with work on living cells.
Genome Instability and Cancer: Biochemical Society Irish Area Section Focused Meeting held at National University of Ireland, Galway, Ireland, 4 December 2008. Organized and Edited by Michael Carty (National University of Ireland, Galway, Ireland).
C.A.H. carried out this work while in receipt of a Ph.D. studentship from the Department of Employment and Learning (Northern Ireland). The work was funded partly by the European Union Framework 6 grant ITS-LEIF (Ion Technology and Spectroscopy at Low Energy Ion Beam Facilities) [grant number RII3/026015].