**7. LEEs interaction and induced damage under cellular conditions**

The gas‐ and condensed‐phase experiments with DNA and its constituents discussed previ‐ ously were performed under ultrahigh vacuum (UHV) conditions to permit use of electron beams and mass spectrometry, and to better control the molecular environment. While such experiments provide information on the direct effects of LEEs, they do not reveal how LEEs can indirectly damage DNA. Comparatively, due to the experimental difficulties related to the production and observation of LEEs in aqueous media, studies on the indirect damage of LEEs to DNA have not been greatly developed.

showed that LEEs are able to effciently decompose this amino acid via DEA and dipolar dissociation (DD), when the molecule is chemisorbed via the SH group to a gold surface.

Regarding the protective effect of amino acids on DNA against LEEs, Solomun et al. [136] reported that the single‐strand DNA‐binding *E. coli* protein can effectively inhibit the forma‐ tion of SSBs by 3‐eV electrons in oligonucleotides. Ptasińska et al. [137] subsequently investi‐ gated by post‐irradiation analysis with HPLC‐UV, the molecular fragmentation induced by 1‐ eV electrons in films comprising the GCAT tetramer and one of the two amino acids, glycine and arginine. At low ratios (*R*) of amino acid to GCAT (i.e., *R* < 1), particularly for glycine, the total oligonucleotide fragmentation yield unexpectedly increased. At higher ratios (1 ≤ *R* ≤ 4), protection of DNA from damage by electrons was observed for both glycine and arginine. Therefore, the amino acid probably reduced electron capture by GCAT and/or the lifetime of the TMA that initiates DEA process. A similar conclusion regarding the stability of the amino acid side chain–nucleobase complexes can be deduced from the theoretical studies of Wang et al. [138]. Wang and coworkers performed calculations at the B3LYP/6‐311G(d,p)‐level anionic hydrogen‐bonded complexes formed between the amino acid side chains and the nucleobase

Furthermore, by studying via first‐principles molecular dynamics simulations a model system composed of thymine and glycine, Kohanoff et al. [139] recently investigated the protection of DNA by amino acids against the effects of LEEs. They considered thymine–glycine dimers and a condensed‐phase model consisting of one thymine molecule solvated in amorphous glycine. These results indicated that at room temperature, the amino acid chemically and physically performs the role of a protective agent for the nucleobase. In a chemical mechanism, the excess electron is first captured by the thymine; then, a proton is transferred in a barrierless way from a neighboring hydrogen‐bonded glycine. Reducing the net partial charge on the thymine molecule stabilizes the excess electron. In the physical mechanism, glycine molecule acts as an electron scavenger to capture the excess electron directly, which prevents the electron to be localized in DNA. Protecting the nucleobase via the latter mechanism requires a predisposition for proton transfer to the oxygen in the carboxylic acid group of one of the involved amino acids. Consequently, raising the free‐energy barrier associated with strand breaks, prompted by these mechanisms, can halt further reactions of the excess electron within the strand of DNA, for instance, transferring the electron to the backbone which leads to induce a strand break in DNA. Increasing the ratio of amino acid to nucleic acid will enhance the protecting role of amino acids, and accordingly will decrease the induction of DNA strand breaks by

**7. LEEs interaction and induced damage under cellular conditions**

The gas‐ and condensed‐phase experiments with DNA and its constituents discussed previ‐ ously were performed under ultrahigh vacuum (UHV) conditions to permit use of electron beams and mass spectrometry, and to better control the molecular environment. While such experiments provide information on the direct effects of LEEs, they do not reveal how LEEs

guanine.

196 Radiation Effects in Materials

LEEs, as shown experimentally [137, 139].

Ideally, to understand how the fundamental mechanisms in LEE–DNA interactions are adapted in living cells, the experimental studies should be extended to the more complex dynamic molecular environment of the cell, or more realistic ones, for the DNA molecule that contains essentially water, oxygen, histones, and DNA‐binding proteins [99]. For instance, in the work of Ptasińska and Sanche [140], the ESD yields of different anions desorbed by 3–20 eV electron impact on GCAT films were measured under an aqueous condition, corresponding to 5.25 molecules of water per nucleotide. Their experiments demonstrated that adding water to dry DNA results in the binding of the molecule to the phosphate group at the negatively charged oxygen [141], and then formation of a complex of tetramer and a water molecule (DNA•H2O). This complex permits the formation of a new type of dissociative core‐excited TMA located on the phosphate group, which decays by O‾ desorption under electron impact via a resonance at 11–12 eV and by OH‾ desorption from breaking the P–O bond. H‾ also desorbs by dissociation of a TMA of the complex which causes bond cleavage on the H2O portion. Moreover, LEE‐induced damage to DNA via DEA enhances by a factor of about 1.6 when an amount of water corresponding to 60% of the first hydration layer is added to vacuum‐ dried DNA. Although the magnitude of this enhancement is considerable, it is still much smaller than the modification in yields of products produced by the first hydration layer surrounding the DNA during the radiochemical events that follow the deposition of the energy of LEE in irradiated cells. Theoretical and experimental studies were concurrently carried out on the diffraction of 5–30 eV electrons in hydrated B‐DNA 5'‐CCGGCGCCGG‐3' and A‐DNA 5'‐CGCGAATTCGCG‐3' sequences by Orlando et al. [142]. They postulated that compound H2O•DNA states may contribute to the modification of strand breaks yield functions [142, 143]. Furthermore, Orlando et al. noted that lowering of the threshold energy for DSBs below 5 eV may be correlated with the presence of these compound states. In this case, an initial "core‐ excited" resonance would autoionize, yielding electronically excited water‐derived states and a low‐energy electron. The electronically excited state dissociates forming reactive O, OH, and H, which can lead to sugar–phosphate bond breakage. The slow electron could moreover scatter inelastically within a limited mean free path and excite a "shape" resonance of a base on the opposite strand. The combination of these two energy‐loss channels could lead to a DSB. This type of DSB requires the presence of water and is difficult to be repaired due to the close proximity of damage sites.

Recent work using graphene‐coated gold thin films also signaled the significance of the existence of water molecules in DNA damage mediated by "shape" resonances [144]. This is likely due to the influence of water on lowering the barrier for charge transfer from the base to the sugar–phosphate bond. In addition, the binding interaction of DNA with graphene allows direct coupling to the phosphates as well as more direct scattering with the guanine and adenine bases. Electrons that have not been captured by DNA bases can be captured by graphene and immediately transferred over 200 nm within < 0.36 ps. The environmental or graphene substrate interactions are critical, and at least two mechanisms occur simultaneously during DNA damage on monolayer graphene: direct base capture and ballistic transfer from the graphene.

An alternative approach to simulate cellular conditions has been recently developed by Alizadeh et al. [145] to investigate LEE‐induced DNA damage under atmospheric conditions and at various levels of humidity and oxygen. Thin films of plasmid DNA deposited on tantalum and glass substrates were exposed to Al K<sup>α</sup> X‐rays of 1.5 keV. The general features of the photo‐ejected SE from the metallic surfaces exposed by primary X‐ray photons are well understood; in particular, more than 96% of SEs emitted from tantalum lie below 30 eV and the energy distribution peaks around 1.4 eV, with an average energy of 5.85 eV [145]. Whereas the damages induced in DNA deposited on glass are due to soft X‐rays, those arising from DNA deposited on tantalum result from the interaction of X‐rays + LEEs. The difference in the damage yields measured in the samples deposited on two different substrates is ascribed to the interaction of LEEs with the DNA and its nearby atmosphere.

Alizadeh and Sanche [146] employed this technique to examine how the presence of several cellular components (such as, O2, H2O and O2/H2O) modulates the LEE‐induced damage to DNA molecules. They observed that for hydrated DNA films in an oxygenated environment, the additional LEE‐induced damage that results from the combination of water and oxygen exhibits a super‐additive effect, which produces a yield of DSB almost seven times higher than that obtained by X‐ray photons. More recently, they reported the formation of four radiation‐ induced products from thymidine by soft X‐rays and LEEs, specifically base release, and base modification including 5‐HMdUrd, 5‐FordUrd, and 5,6‐DHT [147]. Of the products analyzed, thymine release was the dominant channel arising from N‐glycosidic bond cleavage involving *π*\* low‐lying TMA. A LEE‐mediated mechanism was proposed to explain observation of 5‐ HMdUrd and 5‐FordUrd products, which involve loss of hydride (‐H‾) from the methyl group site via DEA. *G*‐values derived from the yield functions indicate that formation of free thymine, 5‐HMdUrd, and 5‐FordUrd are promoted by an oxygen environment rather than a nitrogenous atmosphere, since the numbers and reactivity of radicals and ions are formed via interactions of radiation with O2, and are considerably larger than under N2. Moreover, O2 can additionally react with C‐centered radicals, thereby "fixing" or rendering the damage permanent. In contrast, no 5,6‐DHT was detected when samples were irradiated under an O<sup>2</sup> atmosphere, indicating that O2 molecules react with an intermediate radical compound, thereby inhibiting the pathway for 5,6‐DHT formation [147].

Recently, novel decay mechanisms for electronic excitations and correlated electron interac‐ tions have become subjects of intense study. Just over a decade ago, Cederbaum et al. [148– 150] proposed an ultrafast relaxation process in inner valence levels, which occurs in molecular systems with weakly bound forces, such as van der Waals forces or hydrogen bonding. This mechanism referred to as intermolecular Coulomb decay (ICD) is possible mainly due to the couplings and interactions induced by the local environment. Unlike most ionization proc‐ esses, ICD results in the ejection of an electron from the neighbor of an initially ionized atom, molecule, or cluster [151]. The energy of the ICD electron is low, typically less than 10 eV. ICD is expected to be a universal phenomenon in weakly bound aggregates that contain light atoms and may represent a hitherto unappreciated source of LEEs. Though most ICD measurements have concentrated on rare gas clusters, new sophisticated experimental approaches have detected ICD in large water clusters [152] or at condensed‐phase interfaces containing water dimers and clusters [151].

during DNA damage on monolayer graphene: direct base capture and ballistic transfer from

An alternative approach to simulate cellular conditions has been recently developed by Alizadeh et al. [145] to investigate LEE‐induced DNA damage under atmospheric conditions and at various levels of humidity and oxygen. Thin films of plasmid DNA deposited on tantalum and glass substrates were exposed to Al K<sup>α</sup> X‐rays of 1.5 keV. The general features of the photo‐ejected SE from the metallic surfaces exposed by primary X‐ray photons are well understood; in particular, more than 96% of SEs emitted from tantalum lie below 30 eV and the energy distribution peaks around 1.4 eV, with an average energy of 5.85 eV [145]. Whereas the damages induced in DNA deposited on glass are due to soft X‐rays, those arising from DNA deposited on tantalum result from the interaction of X‐rays + LEEs. The difference in the damage yields measured in the samples deposited on two different substrates is ascribed to

Alizadeh and Sanche [146] employed this technique to examine how the presence of several cellular components (such as, O2, H2O and O2/H2O) modulates the LEE‐induced damage to DNA molecules. They observed that for hydrated DNA films in an oxygenated environment, the additional LEE‐induced damage that results from the combination of water and oxygen exhibits a super‐additive effect, which produces a yield of DSB almost seven times higher than that obtained by X‐ray photons. More recently, they reported the formation of four radiation‐ induced products from thymidine by soft X‐rays and LEEs, specifically base release, and base modification including 5‐HMdUrd, 5‐FordUrd, and 5,6‐DHT [147]. Of the products analyzed, thymine release was the dominant channel arising from N‐glycosidic bond cleavage involving *π*\* low‐lying TMA. A LEE‐mediated mechanism was proposed to explain observation of 5‐ HMdUrd and 5‐FordUrd products, which involve loss of hydride (‐H‾) from the methyl group site via DEA. *G*‐values derived from the yield functions indicate that formation of free thymine, 5‐HMdUrd, and 5‐FordUrd are promoted by an oxygen environment rather than a nitrogenous atmosphere, since the numbers and reactivity of radicals and ions are formed via interactions of radiation with O2, and are considerably larger than under N2. Moreover, O2 can additionally react with C‐centered radicals, thereby "fixing" or rendering the damage permanent. In contrast, no 5,6‐DHT was detected when samples were irradiated under an O<sup>2</sup> atmosphere, indicating that O2 molecules react with an intermediate radical compound, thereby inhibiting

Recently, novel decay mechanisms for electronic excitations and correlated electron interac‐ tions have become subjects of intense study. Just over a decade ago, Cederbaum et al. [148– 150] proposed an ultrafast relaxation process in inner valence levels, which occurs in molecular systems with weakly bound forces, such as van der Waals forces or hydrogen bonding. This mechanism referred to as intermolecular Coulomb decay (ICD) is possible mainly due to the couplings and interactions induced by the local environment. Unlike most ionization proc‐ esses, ICD results in the ejection of an electron from the neighbor of an initially ionized atom, molecule, or cluster [151]. The energy of the ICD electron is low, typically less than 10 eV. ICD is expected to be a universal phenomenon in weakly bound aggregates that contain light atoms and may represent a hitherto unappreciated source of LEEs. Though most ICD measurements

the interaction of LEEs with the DNA and its nearby atmosphere.

the pathway for 5,6‐DHT formation [147].

the graphene.

198 Radiation Effects in Materials

Random damage to cellular biomolecules such as DNA is associated with the onset of cancer, whereas the controlled targeted local release and interactions of LEEs can be used as effective therapeutic cancer treatment agents. Since ICD is a source for the ejection of slow electrons, it has been proposed that ICD could play a role in the induction of SSB and DSB in DNA [153]. Estimation by Grieves and Orlando [152] indicated that ICD may represent up to 50% of the SSB probability for energy depositions >20 eV and ionization events directly at the DNA–water interface. Since the formation of DSBs requires excitation energies >5 eV, the impact on DSBs is expected to be much lower. If ICD contributes significantly to DNA damage, this could be exploited during X‐ray treatment of cancer. **Figure 4** schematically shows that how utilizing of X‐ray interactions with gold nanoclusters within living cells, which subsequently results in releasing both Auger and ICD electrons, has been suggested as a potential strategy for targeted cancer treatment [148].

**Figure 4.** (a) Resonant Auger decay process following X‐ray excitation. A second process known as interatomic or in‐ termolecular Coulomb decay (ICD) can also occur, leading to the ejection of slow electrons and adjacent holes. (b) Pos‐ sible exploitation of Au nanoparticles and ICD in the controlled radiation damage of cells [148].

After such extensive studies on LEE‐induced damage under "near"‐cellular conditions, it was only very recently that the lethal effects of LEEs in cells have been demonstrated by Sahbani et al. [154], who investigated the biological functionality of DNA, via a simple model system comprising *E. coli* bacteria and plasmid DNA bombarded by LEEs. In these experiments, highly ordered DNA films were arranged on pyrolytic graphite surface by molecular self‐assembly technique using 1,3‐diaminopropane ions to bind together the plasmid DNAs [155]. This assembly technique mimics somewhat the action of amino groups of the lysine and arginine amino acids within the histone proteins. These authors measured the transformation efficiency

**Figure 5.** (a) Variation of transformation efficiency of *E coli* by pGEM 3Zf(‐) plasmids irradiated by 0.5–18 eV electrons at a fluence of 27 × 1013 electrons/cm2 . The vertical axis is inverted. Effective yield functions for (b) single‐strand breaks (SSBs), (c) double‐strand breaks (DSBs), and (d) DNA cross‐links [183].

of *E. coli* JM109 bacteria (essentially the number of bacterial colonies grown in an antibiotic environment) after insertion into the cells of [pGEM‐3Zf (‐)] plasmid, which when undamaged, can confer resistance to the antibiotic ampicillin. Before transformation, the plasmids were irradiated with electrons of specific energies in the energy range 0.5–18 eV [156]. Cells receiving severely damaged plasmids will not grow, and the transformation efficiency will be reduced. The loss of transformation efficiency plotted as a function of electron energy is shown in **Figure 5**. It reveals maxima at 5.5 and 9.5 eV, coincident with the maxima observed in the yields of DNA DSBs, which were attributed to the formation of core‐excited TMAs. These results indicated that the effects of TMAs are observable in the electron‐energy dependence of biological processes with negative consequences for cell viability. The result provides further evidence that LEEs play important roles in cell mutagenesis and death during radiotherapeutic cancer treatment [156].
