Enhancement of radiation effect by heavy elements

Mutation Research 704 (2010) 123–131

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Mutation Research/Reviews in Mutation Research journal homepage: www.elsevier.com/locate/reviewsmr Community address: www.elsevier.com/locate/mutres

Review

Enhancement of radiation effect by heavy elements K. Kobayashi a,*, N. Usami a, E. Porcel b, S. Lacombe b, C. Le Sech b a b

Photon Factory, KEK, Tsukuba, Japan Univ. Paris Sud 11, Orsay, France

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 September 2009 Received in revised form 21 December 2009 Accepted 5 January 2010 Available online 13 January 2010

The enhancement of radiobiological effects by heavy elements is reviewed. As an underlying mechanism, Auger effects have been stressed which can be induced via inner-shell photoabsorption or via excitation and/or ionization by secondary electrons. Latter channel of Auger induction expands the applicability of Auger enhancing phenomena to electron and hadron therapy. After discussion on the required characteristics for radiosensitizers, possibility of nanoparticles of Au or Pt is mentioned since they could be synthesized or modified as ideal radiosensitizers. ß 2010 Elsevier B.V. All rights reserved.

Keywords: Radiobiological enhancement Auger effect Heavy elements Nanoparticle Cancer therapy

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Radiation therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Optimization of radiation therapy by various methods . . . . . . . . . . . . . . . . . . . 1.2.1. Concentration of radiation energy, or physical dose, on target tissue 1.2.2. Inhibition of repair processes in cells or tissue . . . . . . . . . . . . . . . . . . Auger effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. What are Auger effects? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. How can we induce Auger effects? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Utilization of radioisotopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Utilization of photons and charged particles . . . . . . . . . . . . . . . . . . . . Enhancement of radiobiological effects by heavy elements. . . . . . . . . . . . . . . . . . . . . . 3.1. Brief history of the biological effect of the photon-induced Auger effect . . . . . 3.2. Results with plasmid DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Results with cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Mechanistic consideration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. Primary physical events and Auger effect . . . . . . . . . . . . . . . . . . . . . . 3.4.2. Processes in aqueous media and the role of water radicals . . . . . . . . 3.4.3. Role of intracellular localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Application to therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Design of sensitizers and drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Possibilities of nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

* Corresponding author. Tel.: +81 29 864 5655; fax: +81 29 864 2801. E-mail address: [email protected] (K. Kobayashi). 1383-5742/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mrrev.2010.01.002

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1. Introduction

order to increase the physical radiation dose, that is, achieve dose reinforcement.

1.1. Radiation therapy Malignant tumors or cancers have now become one of the most frequent causes of death in human society; thus, cancer therapies are attracting much attention. Among various therapeutic methods, radiation therapy has the advantage of noninvasiveness, in contrast to surgical treatment; however, it can lead to the possibility of a secondary tumor. Because of the noninvasive nature of radiation therapy, lesser physiological and psychological burden is placed on patients than compared to that resulting from surgical or pharmaceutical treatments. For these reasons, recovery is better after a radiation therapy than from other protocols. Although it cannot be applied to all types of cancer, radiation therapy is now established as a principal option for treatment, and is thus the subject of intensive study to improve its effectiveness. This article reviews recent studies of radiobiological effects from a mechanistic perspective. Though most of these are basic studies, some ideas obtained from these results should lead to new developments that can be practically applied to therapy in the near future. 1.2. Optimization of radiation therapy by various methods 1.2.1. Concentration of radiation energy, or physical dose, on target tissue The action of ionizing radiation, which includes both photons and particles, is initiated by the transmission of its energy to materials such as living organisms or the human body. It should be noted that X- and gamma-photons deposit most of their energy through secondary electrons that are generated by photoelectric or Compton effects. Fast-charged particles with a high kinetic energy collide with other particles, mainly electrons, and lose kinetic energy. Through these processes, energy is deposited in the matter in the radiation field, stimulating radiobiological effects through complicated reactions that include physical, chemical, and biological processes. In a therapeutic situation, Xrays or gamma-rays are collimated in the shape of the target tissue (cancer tissue) by means of appropriately designed slits, then irradiated to the patient. The radiation energy is delivered to the target tissue, but it is also delivered to the healthy tissue located in front of and behind the target in the beam area. Thus, this leads to detrimental effects on the irradiated healthy tissue as well as the target tissue. Therapeutic irradiation plans must be designed so that the maximum therapeutic effect is delivered to the target tissue, while the damage to the healthy tissue remains at a level that is tolerable for the patient. One of the methods used to achieve this is by using the multiport irradiation method, in which the patient is irradiated from multiple directions. This makes it possible to enhance the irradiation dose in the cancer tissue without delivering intolerable doses to the surrounding healthy tissue. In each irradiation, the beam shape must be carefully adjusted to the shape of the target to which the beam is projected. Another method to increase the dose delivered to the target tissue is to use elements that have large photoabsorption cross sections. This method is based upon the nature of elements and the energy of the incident X-ray photons. In theory, in the presence of highly absorbing elements in tumors, X-ray photoabsorption in the tissue can be effectively enhanced by tuning the energy of the X-rays to that of the inner-shell absorption edge of the targeted elements. As detailed below, Auger deexcitation processes take place after photoionization and induce the instantaneous emission of Auger electrons, which are responsible for the enhancement of the dose delivered to the tissue. As a result, superior radiation effects can be expected when the tissue contains such elements. These two methods are combined in

1.2.2. Inhibition of repair processes in cells or tissue It is well known that radiobiological effects are modified by the chemical and biological conditions of the cell, and that the integrated effects are the result of complex chemical and biological reactions. Among these processes, the final and most important mechanism is the process of cellular repair, in which the cell repairs the DNA damage produced by radiation. This DNA repair system has been well studied. It has been found that (a) various types of DNA damage caused by radiation can be repaired; (b) quantitatively speaking, most DNA damage, including breaks, is repaired, and unrepaired or mis-repaired damage is considered to be the cause of cell death or genetic changes; and (c) the repair system is essential for living cells to survive in oxidative circumstances since oxidative DNA damage is constantly produced by oxygen radicals generated in the physiological process of energy production. On the basis of these results, we can expect to achieve an enhancement of radiobiological effects by lowering the repair efficiency. The selective production of a less repairable type of damage or the inhibition of a repair mechanism would affect the repair efficiency. Less repairable forms of DNA damage are considered to be produced by the radiation of high linear energy transfer (LET) – energy deposited along the track defined in terms of keV/mm, such as heavy ion particles. This type of radiation produces energy deposition events that are very densely positioned along the track, and hence, produces multiple DNA damages in localized areas along the track. This type of damage, which is called clustered DNA damage, is believed to be less responsive to repair mechanisms and is a major contributory factor to the induction of biological defects. On this basis, high LET radiation is known to produce more pronounced radiobiological effects than low LET forms of radiation such as X-rays. It should be noted that Auger processes, which follow photoionization processes in matter (see details below), also contribute to the enhancement of the density of the energy deposition events around photoabsorption sites. 2. Auger effects 2.1. What are Auger effects? Negatively charged electrons usually occupy well-defined orbits, or shells, around the positively charged nucleus. The binding energies of electrons, or in other words, the energy necessary to liberate electrons from their bound state, is quantified and depends upon the positive charge, or the atomic number, of the nucleus. The orbits or shells can accommodate a finite number of electrons. These shells are called K, L, M, N, and O. The number of electrons that can be accommodated in each shell is 2 for a K shell, 8 for L, 18 for M, and 32 for N at a maximum. The electrons in the K shells are the most tightly bound to the nucleus. Usually, the atoms or molecules stay in the lowest energy state as a whole at room temperature. When an energy higher than the binding energy of the inner shell is applied to an atom or a molecule (for instance, by photoabsorption), the system is ionized. That is, an electron is ejected and a vacancy is left in the shell (inner-shell ionization). This ionization is followed by a process of deexcitation in which an electron of an outer shell (higher energy state) drops into the lower energy shell in order to fill the vacancy. The energy difference between the two shells is thus transferred to a fluorescence photon or to another electron that is ejected from an outer shell. The latter phenomenon is called the Auger effect, and the ejected electron is called an Auger electron. As a result, one vacancy in the inner shell is converted into two vacancies in an outer shell. If another outer

K. Kobayashi et al. / Mutation Research 704 (2010) 123–131 Table 1 Average yields of Auger electrons and localized energy deposition for some radionuclides (from Sastry et al. [1]). Radionuclide

Average yield per decaya

Energy (eV) deposited in a 5-nm sphere

Cr-51 Fe-55 Ga-67 Se-75 Br-77 Tc-99m In-111 I-125 Pt-193m Pt-195m Tl-201

6 5 5 7 7 4 8 19 27 33 20

210 240 260 270 300 280 450 1000 1800 2000 1400

a

(5) (3) (3) (5) (5) (4) (7) (17) (23) (27) (17)

Low energy electron (<1 keV) yield is given in parentheses.

shells exist in the atom, these two vacancies can be filled with other electrons and a succession of further Auger effects takes place, in what is called an Auger cascade. The number of emitted electrons, which is multiplied until the outermost shell is reached, is larger in high-Z atoms. For example, the ionization of the K shell of platinum leads to the emission of approximately 30 electrons. The calculated average yield of Auger electrons for the decay of some radionuclides via electron capture is tabulated in Table 1 [1], and the calculated ranges of low energy electrons in water are shown in Table 2 [2]. Therefore, the presence of platinum in matter enhances the number of electrons emitted around the emitter. As a result, the energy deposition due to the ionization events induced by secondary (Auger) electrons occurs very densely around the atom. The amount of energy deposited around the Auger decay site is also shown in Table 1. From these values one can easily calculate the enormous value of the absorbed dose in Gy in the 5-nm sphere. In the case of X-ray irradiation, it should also be noted that (1) the photoabsorption cross sections of inner-shell electrons are much larger than those of the outer shell electrons [3], and (2) the total photoabsorption cross section is larger for high-Z elements than for low Z elements [4]. This indicates that in most cases, X-ray photoabsorption not only produces photoelectrons, but it also induces Auger effects (and Auger electrons) at the photoabsorption site. In conclusion, the presence of high-Z elements produces enhanced radiobiological effects as efficiently as high LET radiation, even with X-ray photon irradiation.

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2.2.2. Utilization of photons and charged particles A vacancy can be produced in an inner shell with photons or charged particles, including electrons. In the case of photons, when the energy is higher than the inner-shell binding energy of the target atom, an electron can absorb the photons via a photoelectric effect, and a vacancy is thus produced. The probability of a photoelectric effect taking place depends on both the photon energy and the targeted element. The energy dependence of the photoabsorption cross section is called the ‘‘absorption spectrum.’’ The ionization of a specific instance of inner-shell ionization is effectively induced by choosing the proper energy of the X-rays. For this purpose, synchrotron radiation is the only light source that gives access to intense monoenergetic photon sources. As described above, Auger effects are induced very frequently in Xray irradiated samples, even though X-rays are not monochromatized. Collisions with high-energy charged particles can also produce inner-shell ionization. Electrostatic interactions between the incident charged particle and the orbital electrons produce enough energy to excite or ionize inner-shell electrons. The ionization of an inner shell is thus followed by Auger deexcitation and the emission of Auger electrons. 3. Enhancement of radiobiological effects by heavy elements Radiobiological effects are usually compared on the basis of the absorbed dose (1 Gray (Gy) = 1 Joule absorbed per kg of matter). The term relative biological effectiveness (RBE) is used to quantitatively characterize the effect that radiation has upon biological matter. RBE is defined as the inverse ratio of the dose that a new protocol requires in order to obtain the same radiobiological effect as that obtained from a standard source. Gamma-rays are usually used as the standard radiation source. The RBE is equal to 1 when the dose required to produce a certain effect is equal to the dose of gamma-rays that can produce that same effect. High-energy heavy particle radiation generated by accelerators is well known for exhibiting a high RBE. The RBE of carbon atoms used in hadrontherapy is approximately 4. Several therapeutic centers for cancer that use heavy ion accelerators are currently in operation around the world and have succeeded in obtaining good results. This review focuses on the radiobiological enhancement induced by heavy elements that are artificially incorporated into cells or tissues.

2.2. How can we induce Auger effects? Auger effects, or Auger cascades, originate from inner-shell ionization. The ionization of the inner shells in atoms can be observed or induced in the following situations. 2.2.1. Utilization of radioisotopes Some radioisotopes decay in a specific way such that an electron in the innermost K shell is absorbed in the nucleus, which results in a decrease in the atomic number (one proton becomes a neutron). The vacancy in the K shell produced by the electron capture is filled with an L shell electron. An Auger effect is thus induced. Table 2 Range of low energy electrons in continuous slowing down approximation (compiled from Watt [2]). Electron energy (eV)

Rangecsda in water (nm)

50 100 200 500 1000

2.5 4.8 9.0 23 58

3.1. Brief history of the biological effect of the photon-induced Auger effect The photoabsorption spectra in the X-ray region have long been recognized by biophysicists as well as physicists. In the X-ray region, the K shell, among all other shells, is known to have the largest cross section. Investigations and applications at this energy level have failed, however, due to the polychromatic nature of Xrays from X-ray tubes. In 1993, Laster et al. [5] proposed photon activation therapy (PAT) as a promising form of cancer therapy. In this technique, heavy element-containing molecules are administered to the patient, and monochromatic X-rays tuning to the K shell absorption edge of the heavy element are irradiated by using synchrotron radiation as a light source. Before their work, a Japanese radiobiology group led by T. Ito initiated a spectroscopic study of radiobiological effects by constructing a dedicated beam line in an INS-SOR ring in Tokyo [6]. In 1983, the Photon Factory, equipped with a 2.5 GeV electron storage ring, was commissioned; it extended their ability to work in the X-ray region. They succeeded in experimentally demonstrating the enhancement of radiobiological killing effects by the Auger effect for phosphorus in

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Fig. 1. (A) Absorption spectrum of DNA film around K-shell absorption edge of phosphorus. The dotted line is the total photoelectron yield of KH2PO4. The three vertical arrows indicate the X-ray energies used for the irradiation experiments shown in panels (B) and (C). (B) Lethal effect and (C) induction of genetic changes in yeast cells irradiated with X-rays tuned to resonance absorption peak (2.153 keV (triangle)) and both sides of the peak (2.147 eV (square) and 2.160 keV (circle)) [9]. Unit of ‘‘C/kg’’ in SI is used to show X-ray exposure, which is equivalent to ‘‘R (Roentgen)’’ in cgs units.

DNA, as shown in Fig. 1. Their research has been reviewed in the references [7,8]. 3.2. Results with plasmid DNA Inner-shell photoabsorption by phosphorus has attracted much attention for two reasons: (1) inner-shell photoabsorption is followed by the Auger effect, which emits Auger electrons such that a multiply charged atom or molecule is produced; and (2) phosphate constitutes the main chain of the DNA molecule together with deoxyribose. It is also of practical importance that the absorption cross section at the resonance of K shell is about 5 times larger than the off-resonance K-shell absorption cross section [9]. The enhancement by phosphorus in the induction of single and double strand breaks was clearly observed [10]. In other works, the Auger enhancement by a bromine atom, which was incorporated in phage DNA as bromodeoxyuridine, was studied by Furusawa et al. [11]. They showed that an artificially introduced bromine atom can enhance radiobiological effects as a consequence of K-shell ionization followed by Auger effects. Recently, Le Sech et al. [12,13] demonstrated that a platinum-containing molecule acts as a ‘‘radiosensitizer’’ when photons corresponding to the absorption edge of the inner shell of platinum are used. A platinum compound, chloroterpyridine platinum (PtTC), was mixed with plasmid DNA with a ratio of 1 Pt atom per 10 nucleotides, and the mixed sample was irradiated with monochromatic X-rays tuned to the resonant photoabsorption energy of the L(III) shell of the platinum atom. Although the atomic abundance of Pt in the sample was very small, the yield of double

strand break (DSB) of DNA was higher, with X-rays above the absorption edge than below the edge. This gave rise to a new understanding that nonphysiological molecules can act as a sensitizer if they are incorporated into cells. Le Sech et al. also reported that the ratio of DSB to single strand break (SSB) increases upon on-resonance photoabsorption, suggesting that damage that is more severe to living cells, DSB, is favored in the presence of Auger effects. These works were done with dehydrated dry samples, but successive works showed that the amplification effect of high-Z atoms is also observable in solution [14,15]. 3.3. Results with cells The biological effects induced by monochromatic photons have been investigated on a cellular scale, anticipating the possibility of the application of photon activation therapy as a medical tool. The first data on the enhanced killing of cells prelabeled with 5bromodeoxyuridine were reported for mammalian cells by Shinohara et al. [16], and for E. coli by Maezawa et al. [17]. Similar works were done using a mammalian cell line sensitized with 5bromodeoxyuridine [18] and with 5-iododeoxyuridine [5] by Laster et al. Usami et al. [19] discussed the enhancement achieved by the incorporation of bromine into DNA from the viewpoint of the absorbed energy, or dose, using the substituted fraction of thymine with bromo-uracil. They concluded that the enhancement could not be explained solely by the increased absorbed energy, or dose, and that some damage specific to the Auger cascade might have been produced. Such Auger-induced DNA damage was suggested to be less repairable, cluster type damage [19] similar

K. Kobayashi et al. / Mutation Research 704 (2010) 123–131

to the damage induced by high LET heavy particles. This idea was supported by the results obtained by Maezawa et al. [20], which showed that the chromosomal damage produced by the innershell ionization of phosphorus was not repaired at all, while the damage done by X-rays, which never induce inner-shell ionization of phosphorus, was repaired to one-third of the produced ones by the action of the cellular repair system. Jonsson et al. [21] tried to find the enhancement by inner-shell ionized indium atoms incorporated into the cell. No significant difference in the degree of survival between irradiations above and below the K-edge could be observed, due to the small amount of indium incorporated into the cell. This demonstrates the importance of selecting the chemicals or methods used to deliver the target atoms into the cell, in order for photon activation therapy using heavy elements to become effective. 3.4. Mechanistic consideration 3.4.1. Primary physical events and Auger effect To further improve the enhancing properties and applicability to therapy of photon activation therapy using heavy elements, it is crucial to review the entire range of radiobiological phenomena, from the molecular dynamics that follow the interaction of radiation with matter, to the enzymatic reactions which have substantial biological effects. When X-ray photons interact with matter, the first mechanism that is activated is either photoelectric absorption or a Compton effect. In photoelectric absorption, an X-ray photon is absorbed by an atom and, using the energy of the photon, an electron is emitted, leaving an ionized atom or molecule. When an electron from an inner shell is ejected, this ionization is followed by an Auger deexcitation process as already mentioned. It is known that the absorption cross section is larger in the inner shells than in the outer shells. The Compton effect is a nonelastic scattering of an X-ray photon by the orbital electron in the atom, which causes the emission of an electron (Compton electron). This also leaves the atom or molecule in a positively charged state. The scattered photon loses an energy which is equal to the sum of the kinetic energy of the Compton electron and the binding energy of the ejected electron. These ionization events of molecules correspond to the energy deposition events in the matter. More importantly, the electrons emitted either by photoelectric effects or by Compton effects can further induce energy deposition in matter until these ionization events totally exhaust their kinetic energy. The range of energetic Compton electrons might be up to several hundred microns. In cases of lower energy, of photoelectrons or Auger electrons, however, the range of size becomes much smaller; for instance, the range of 1 keV electrons is calculated as around 0.05 mm. Energy deposition events are produced along these tracks, and radiation energy is thus distributed in the biological system. These physical events initiate biophysical processes in the irradiated system. For this reason, radiobiological phenomena are assessed on the basis of the energy deposited in the biological system, and hence, the metric Gy (Gray) is defined as the energy (J) deposited divided by the mass (kg) of the system concerned. However, this quantity corresponds to an averaged value in the target, and is not a sufficient basis upon which to predict the resulting radiobiological phenomena. This point can be more clearly understood if one considers the energy necessary to raise the temperature of 1 g of aqueous solution by just 1 8C. Simple calculation shows that the deposited energy, 1 cal, in 1 g is equal to about 4200 Gy. This value is three orders of magnitude higher than the lethal dose for mammalian cells. In radiation biology, it is important to relate the radiation energy deposited by these physical processes to the resulting radiobiological phenomena from the viewpoint of the amount of energy deposited per event,

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as well as the spatial distribution of these events along the track of the charged particle. In order to characterize the spatial distribution of an energy deposition event in matter, the LET is often used as an indicator. The LET of heavy particles is known to increase with a decrease in the particle energy. For this reason, the Bragg peak appears in the dose distribution profile at the end of the particle track. This parameter is conventionally used for characterization of the heavy particle beam, and biological efficiency is often discussed from the viewpoint of the LET. It has now become a common understanding that high LET heavy particles produce greater biological effects than low LET ionizing radiations, when compared on the basis of the absorbed dose in Gy. On a microscopic scale, this is considered to be the result of the high density of the energy deposition event along the track. Strictly speaking, however, even though the LET values of different particles are the same, the radial distribution of energy deposition events through the secondary electrons, called ‘‘penumbra,’’ depends upon the particle. In the case of electrons, low energy electrons (<1 keV) are known to leave their energy in higher density along their tracks and to exhibit a high LET property [22]. Each electron track, therefore, contains high LET components at its track end. When an Auger cascade is induced, many low energy electrons are emitted from the same atom; hence, the energy deposition density around the site is multiplied by the number of low energy electrons. The relationship between deposited energy and the resulting molecular changes in DNA was studied by Hieda et al. using synchrotron radiation [23,24]. They controlled the energy deposition per event by changing the photon energy from 5 eV to more than 1 keV. They showed that the yields of strand breaks and base damage strongly depend upon the energy deposition per event in the low energy region, and that the yield of double strand breaks (DSB) of plasmid DNA increases more rapidly than single strand breaks (SSB) with an increase in the photon energy. In the region below 20 eV, this energy dependence was also investigated by means of electron bombardment [25]. This approach revealed that resonant dissociative electron attachment, which corresponds to the decomposition of molecules due to electron attachment, plays an important role in producing molecular damage below the level of ionization potential energy. As recently reviewed by Lacombe and Le Sech [26], physical processes induced by low energy ions may also induce damage in biological molecules. As shown in their last work on dry DNA, photons below 60 eV also induce damage efficiently [27]. The effect of high deposition density with electrons was also reported by Tomita et al. [28]. They produced a shower of quasimonoenergetic electrons in aqueous solution using monochromatic synchrotron X-rays, and measured the yields of SSB and DSB in DNA. They found that the yield of DSBs increases with a decrease in the photon energy and, subsequently, with a decrease in the energy of the monoenergetic electron beam. They suggested that the low energy electrons deposit energy more densely than the high-energy electrons. Based on these works, the enhancement of the radiobiological damage induced by Auger cascades can be easily understood. It can thus be predicted that this enhancement of molecular damage is closely related to the photoabsorption spectra of the sample, as evidenced by our works on dry state DNA [10–12]. 3.4.2. Processes in aqueous media and the role of water radicals Secondly, we need to consider the processes in solution that follow the events of energy deposition. The yields of molecular damage in an aqueous system which lead to radiobiological effects were studied with a focus on Auger effects. Due to the atomic abundance in an aqueous system, oxygen in a water molecule has the largest absorption cross section. A large amount of water

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radicals such as OH radicals are produced in the system and they can attack biological molecules, resulting in oxidized molecular changes or damage. This type of damage produced by the action of radicals is called ‘‘indirect damage,’’ while the damage or molecular change produced directly by the interaction with photons or charged particles is called ‘‘direct damage.’’ Due to the large number of radical reactions, molecular damage that is specifically due to fast Auger processes may not be detectable or observable. We succeeded in observing the Auger effect on a phosphorus atom in DNA in aqueous media [29] by showing that the photon energy dependence of the molecular damage yield is very similar to the photoabsorption cross section of the solute sample, reflecting the impact of Auger effects. It should also be mentioned that enhancement can be observed in the presence of dimethyl sulfoxide (DMSO), a specific scavenger of OH radicals [30]. This suggests that enhancement could be observed in conditions in which there is a high rate of scavenging of radicals, such as the intracellular environment. Even more interestingly, a detailed study of photon energy dependence on Pt loaded DNA showed that enhanced damage could be observed when the system is irradiated with X-rays with energies that are higher, and also lower, than the absorption edge of the L-III shell, as shown in Fig. 2 [15]. Since this observation cannot be explained solely by the process of inner-shell photoabsorption followed by Auger effects, it gives rise to a new hypothesis, namely that the inner-shell ionization in Pt atoms may be effectively induced by energetic secondary electrons produced by X-rays in water. The energy deposition by secondary electrons in platinum occurs through electron–electron collision processes. Due to the large number of electrons in a Pt atom, collisions are expected to occur more frequently than in the light elements that constitute biological molecules, hence Auger cascades in platinum become observable. We can thus consider a scheme in which platinum, a heavy element, absorbs the energy of incident secondary electrons. This hypothesis was also tested with heavy particle radiation in the same biological system, namely DNA loaded with a Pt compound in aqueous solution. It was found that DNA strand breaks increase significantly when platinum is present, indicating a radiosensiti-

Fig. 2. (A) Relative excess of the yield of DSB in the presence of PtTC (m(DSB, Pt)) normalized with the values in the absence of PtTC (m(DSB) = m) is plotted against the energy of the irradiated monochromatic X-ray photon. (B) Calculated absorption spectrum of PtTC around the L-III absorption edge of platinum. [15].

Fig. 3. A scheme to illustrate the role of platinum in the mechanism by which DNA damage induction is enhanced in an aqueous system. Briefly, a secondary electron strikes an inner-shell electron in Pt, followed by an Auger cascade. The emitted low energy electrons produce OH radicals densely around Pt causing stand breaks in the DNA.

zation by the high-Z atoms [30]. In particular, it was shown that the main contribution to the induction of DNA damage comes from the action of radicals. This radiosensitizing efficiency, or enhancement, was measured for different incident beams with different LET values [31]. The induction of SSBs and DSBs, as expressed in the number of lesions per Gray, decreases with increasing LET. This is commonly attributed to the recombination of hydroxyl radicals in the tracks and/or to the decrease in heavy particle tracks to deliver the same dose. More interestingly, the efficiency of platinum compounds decreases with increasing LET as well. This shows that the efficiency of platinum may be related to the number of tracks in the medium. These results suggest more a general applicability of a heavy element sensitizer to radiotherapy, in which some synergism between radiotherapy and chemotherapy can be expected. A scheme to illustrate the role of platinum in the enhancing mechanism of DNA damage induction in an aqueous system is shown in Fig. 3. 3.4.3. Role of intracellular localization In most of work done with sensitizers, the focus has been upon the characteristics of their binding to DNA or being included in DNA as substitutes, since it can be expected that the absorbed energy of radiation may be efficiently transferred to DNA [9,11,12]. This transferred energy is what causes the production of DNA damage. Electron transfer or hole transfer may also take place as one of the deexciting processes that originates from the Auger effect or ionization/excitation by radiation [25]. It should be noted that an Auger cascade leaves a multiply ionized molecule that will be neutralized through electron transfer from the neighboring molecules. In aqueous systems that include living cells, the indirect effects mediated by radicals are known to play an important role in the production of DNA damage, as described above. These radicals can react with various molecules in an aqueous solution due to their high reaction coefficients. This determines the diffusion lengths of radicals, or the lifetime of radicals. Diffusion length can be calculated as several 10 of nm in intracellular circumstances in which many kinds of biomolecules are dissolved. These considerations suggest that energy deposition events and the consequent Auger effects need to occur very close to biologically important molecules such as DNA. One important experiment was performed with radioisotopelabeled molecules, whose radioisotopes decay in an electron capture mode, inducing Auger cascades [32,33]. One of the two molecules used in this experiment could bind to DNA in a cell, and the other could not. It was clearly demonstrated that the DNA-binding molecule showed a higher efficiency than the other molecule. These results also indicate that the localization in cells is very important for determining the sensitizing properties of chemicals. It has generally been believed that localization in the nucleus gives higher sensitization due to the higher resulting efficiency in producing

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principle is not sufficient for targeting cancer because FU also accumulates in other actively dividing tissues in the body that are essential to life. Though the accumulation of FU in other tissues is known to cause side effects, it is not easy to synthesize heavy element-containing, nontoxic compounds which can be incorporated specifically into the target cells. Another method, then, would be one that is used in radioimmunotherapy, in which Auger emitters are conjugated with antibodies and administered to the patient. Depending upon the type of antibody used, the chemical sits on the membrane or inside the cell in the target tissue [37]. The encapsulation of the drug in small microspheres may enable it to be delivered to the target tissue by coating the tissue- or the cellspecific antibodies. Cis-platinum, which is a well-known chemotherapeutic agent that shows cytotoxicity, might be used as sensitizer since it is expected to show some degree of synergism between radiotherapy and chemotherapy [38]. However, in designing new chemical formulas for sensitizers, nontoxicity or low toxicity to cells and to the human body is considered to be the top priority. 4.2. Possibilities of nanoparticles

Fig. 4. Cell surviving fractions versus the irradiation dose of carbon ions at LET = 13 keV/mm with PtTC (filled circle) or without PtTC (open circle), and at LET = 70 keV/mm with PtTC (filled triangles) or without PtTC (open triangles) [34].

DNA damage. Recently, Usami et al. [34] irradiated mammalian cells loaded with a Pt-containing molecule, Pt-terpyridine chloride (Pt– TC), whose sensitizing properties had already been proved on DNA in solution (Fig. 4). The sensitizing properties of the Pt compound were thus demonstrated on the scale of living cells. However, it was found by nano secondary ion mass spectroscopy (nano-SIMS) analysis that the chemical was located in the cytoplasm. These results suggest that ultimately, the killing of cells by radiation might be caused by energy deposition in the cytoplasm. The mechanism of cell death as a cytoplasmic event is now one of the hot topics in radiation biology [35]. Wu et al. demonstrated that the irradiation of cytoplasm with alpha particles induces cell death and mutation in a mammalian cell line [36]. Considering the high degree of scavenging activity in cytoplasm, a dense energy deposition around a high-Z atom, presumably due to Auger effects, produces water radicals that are densely positioned around the atom, which could cause some lethal damage in the cytoplasm near to the atom. The mitochondria and the raft structure in the membrane system are proposed as the lethal targets. 4. Application to therapy 4.1. Design of sensitizers and drug delivery Although most of the many works mentioned above are basic studies, they indicate that molecules containing high-Z elements might act as sensitizers. When chemicals are applied to radiotherapy, they must be delivered to the target tissue in order to keep the healthy tissue unsensitized. The important question is how specific the action of a given compound can be to the target tissue. In choosing sensitizers, we need to think of the variety of forms of cancer tissue, as well as the modification that chemicals undergo due to metabolism. The therapeutic drug is generally injected into a vein and then circulates through the blood flow. It is known that chemicals are incorporated into target tissue according to several specific principles. One of these principles is the fact that cancer tissue has a high dividing activity that requires a large amount of DNA precursors. This is why Fluorouracil (FU), which is commonly used in chemotherapy, is incorporated into cancer tissue. This

Recent advances in nanochemistry have paved the way for new strategies for the development of efficient sensitizers. Such strategies make use of nanoparticles, whose size ranges from a few nm to 100 nm, and which are produced by various methods. In order to stabilize them in an aqueous solution, they are usually coated with hydrophilic moieties. Through the choice or modification of these moieties, it becomes possible to control the affinity or specificity of particles to the target molecules, cells, or tissues. Nanoparticles of gold or platinum can be used as sensitizers since they contain many high-Z atoms, and their surfaces can be modified so that they will bind to specific target cells or tissues. Dose enhancement by gold nanoparticles has been simulated by Zhang et al. [39]. They showed that such enhancement is of practical value in a therapeutic situation. Carter et al. [40] calculated microscopic dose distribution from the surface to a distance of 3-nm from a gold nanoparticle in the X-ray irradiation field, and showed that the energy deposition on a nanoscale is larger in the range of a few nm from the surface of the particle, due to low energy Auger electrons escaping from the particle, than the average energy deposition in the bulk solution. This suggests that Auger electrons contribute to the enhancing reactions which take place very close to the surface of the particle. This dynamic was confirmed by their experiments on the enhancement of DNA damage yield in different scavenging environments. The sensitizing properties of nanoparticles have been tested on DNA, living cells, and animals. Different works have confirmed the enhanced induction of strand breaks in X-ray [41] and electron [42] irradiated mixtures of gold nanoparticles and DNA. The application of the sensitizing properties of nanoparticles to the cellular system has been shown on a cancer cell line [43]. Reports from other researchers have also indicated the potential of the nanoparticles as a radiosensitizer [44–47], clearly foreseeing the applicability of these new compounds to radiotherapy. Although direct evidence of the contribution of Auger effects is not presented in these studies, it may be reasonable to make two assumptions. First, that dense ionization, which produces water radicals at a high density around nanoparticles, is due to the low energy electrons that escape from the particles, as shown by Carter et al. [40]. Second, that densely produced water radicals attack some lethal targets located near the particle, since radicals that are sparsely produced by energetic electrons are less effective in producing biologically significant damage. Although nanoparticles may not be incorporated into cell nuclei, they could contribute to the killing of cells by attacking cytoplasmic targets.

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Due to advances in accelerator technologies, hadron therapy facilities are now more accessible. Heavy particle irradiation presents strong advantages in cancer therapy. The ions have a more confined dose profile within the patient and thus a higher biological efficiency in a well-defined volume at the end of the track. When combined with appropriate sensitizers such as nanoparticles, we can expect a higher cure rate from cancer therapy. Research in this regard, with an emphasis on the development of new nanoparticles, is currently being pursued in various laboratories worldwide. 5. Summary This article presents a review of the enhancement of radiobiological effects by heavy elements. We have focused on the underlying mechanism of Auger effects, which can be induced via inner-shell photoabsorption or via excitation and/or ionization by secondary electrons. This latter channel of Auger induction expands the applicability of Auger enhancement phenomena to electron and hadron therapy. Following a discussion on the required characteristics for radiosensitizers, the possible use of nanoparticles of Au or Pt is considered, since their synthesis or modification might enable them to function as ideal radiosensitizers. Conflicts of interest The authors declare that there are no conflicts of interest. References [1] K.S.R. Sastry, R.W. Howell, D.V. Rao, V.B. Mylavarapu, A.I. Kassis, S.J. Adelstein, H.A. Wright, R.N. Hamm, J.E. Turner, Dosimetry of Auger emitters: physical and phenomenological approaches, in: K.F. Baverstock, D.E. Charlton (Eds.), DNA Damage by Auger Emitters, Taylor & Francis, 1988. [2] D.E. Watt, Quantities for Dosimetry of Ionizing Radiations in Liquid Water, Taylor & Francis, 1996. [3] J.H. Scofield, Theoretical Photoionization Cross Sections from 1 to 1500 keV, Lawrence Livermore Laboratory, University of California, 1973 UCRL-51326. [4] B.L. Henke, E.M. Gullikson, J.C. Davis, X-ray interactions, Atomic and Nuclear Data Tables 54 (1993) 181–342. [5] B.H. Laster, W.C. Thomlinson, R.G. Fairchild, Photon activation of iododeoxyuridine: biological efficacy of Auger electrons, Radiat. Res. 133 (1993) 219– 224. [6] T. Ito, Vacuum ultraviolet photobiology with synchrotron radiation, in: R.M. Sweet, A.D. Woodhead (Eds.), Synchrotron Radiation in Structural Biology, Plenum Publishing Corporation, 1989, pp. 221–241. [7] K. Kobayashi, Radiobiology using synchrotron radiation, in: E. Bruttini, A. Balerna (Eds.), Proceedings of the International School of Physics ‘Enrico Fermi’, vol. Course CXXVIII, IOS Press, Amsterdam, 1996, pp. 333–352. [8] K. Kobayashi, Photon-induced biological consequences, in: A. Mozumder, Y. Hatano (Eds.), Charged Particle and Photon Interactions with Matter, Mercel Dekker Inc., 2003, pp. 471–489. [9] K. Kobayashi, K. Hieda, H. Maezawa, Y. Furusawa, M. Suzuki, T. Ito, Effects of Kshell X-ray absorption of intracellular phosphorus on yeast cells, Int. J. Radiat. Biol. 59 (1991) 643–650. [10] K. Hieda, T. Hirono, A. Azami, M. Suzuki, Y. Furusawa, H. Maezawa, N. Usami, A. Yokoya, K. Kobayashi, Single- and double-strand breaks in pBR322 plasmid DNA by monochromatic X-rays on and off the K-absorption peak of phosphorus, Int. J. Radiat. Biol. 70 (1996) 437–445. [11] Y. Furusawa, H. Maezawa, K. Suzuki, Enhanced killing effect on 5-bromodeoxyuridine labelled bacteriophage T1 by monoenergetic synchrotron X-ray at the energy of bromine K-shell absorption edge, J. Radiat. Res. (Tokyo) 32 (1991) 1–12. [12] C. Le Sech, K. Takakura, C. Saint-Marc, H. Frohlich, M. Charlier, N. Usami, K. Kobayashi, Strand break induction by photoabsorption in DNA-bound molecules, Radiat. Res. 153 (2000) 454–458. [13] C. Le Sech, K. Takakura, C. Saint-Marc, H. Frohlich, M. Charlier, N. Usami, K. Kobayashi, Enhanced strand break induction of DNA by resonant metal-inner shell photoabsorption, Can. J. Physiol. Pharmacol. 79 (2001) 196–200. [14] K. Takakura, K. Kobayashi, D.N.A. Degradation, Atomic target method using synchrotron radiation, in: J.C. Salamone (Ed.), Polymeric Materials Encyclopedia, vol. 3, CRC Press, Boca Raton, 1996, pp. 1926–1931. [15] K. Kobayashi, H. Frohlich, N. Usami, K. Takakura, C. Le Sech, Enhancement of Xray-induced breaks of DNA when bound to molecules containing platinum—a possible application to hadrontherapy, Radiat. Res. 157 (2002) 32–37.

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