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1 Radiation Protection Dosimetry Vol. 99, os 1 4, pp () uclear Technology Publishing MECHAISMS F LW EERGY ELECTR DAMAGE T CDESED BIMLECULES AD DA L. Sanche Groupe des Instituts de Recherche en Santé du Canada en Sciences des Radiations Faculté de Médecine, Université de Sherbrooke Sherbrooke, Québec, Canada, J1H 54 IVITED PAPER Abstract It has recently been shown that 3 ev electron impact on vacuum-dry samples of plasmid DA induced substantial yields of single and double strand breaks (SSBs and DSBs). These results are summarised in the present article along with those obtained from the fragmentation of elementary components (i.e. condensed H, bases and sugar analogues) of DA induced by low energy electron impact under ultra high vacuum conditions. By comparing the results from these experiments, it is possible to determine fundamental mechanisms by which low energy electrons damage DA. The decay of transient anions formed on the DA s basic components is found to play a crucial role in producing SSBs and DSBs. Since a large portion of the energy deposited by ionising radiation first leads to the production of low energy secondary electrons, these findings provide basic knowledge necessary to understand the genotoxic effects of high energy radiation and eventually modify these effects at the molecular level. ITRDUCTI Many investigations during the past century have been devoted to the understanding of the alterations induced by high energy radiation in biological systems, more particularly within living cells and the DA molecule. The biological effects of such radiation are not produced by the mere impact of the primary quanta, but rather, by the secondary species generated along the radiation track (1). As these species further react within irradiated cells, they can cause mutagenic, genotoxic and other potentially lethal DA lesions ( 4), such as single and double strand breaks (SSBs and DSBs). Secondary electrons with energies below ev are the most abundant of the secondary species produced by the primary interaction (5 7). For example, a 1 MeV primary photon or electron generates about secondary electrons when its energy is deposited in biological matter (5,6,8). It is therefore crucial to determine the action of secondary electrons within cells, particularly in DA where they could induce genotoxic damage. To investigate such damage induced to vital cellular components, experimental efforts have been devoted to the isolation of biomolecules as thin multilayer films in ultra high vacuum (UHV), where they could be bombarded with a beam of low energy electrons (LEEs) (9 1). Such environmental conditions are necessary to avoid LEE interaction with small surface impurities, which could modify the probed damage. In these experiments, fragments of biomolecules induced by LEE bombardment can be either analysed in situ or outside UHV. In the former, the masses of ions and neu- Contact author Isanche@courrier.usherb.ca tral species desorbing from a multilayer film target are analysed during bombardment (11,1) or the products remaining in the film are characterised by X ray photoelectron spectroscopy after bombardment (13). If sufficient degraded material is produced, the film target can be extracted from the UHV chamber and the products analysed by standard methods of chemical identification (9). In this article, some of these methods are briefly described and recent results obtained from experiments performed with these techniques on the DA molecule and some of its elementary constituents are reviewed. RESULTS FRM LW EERGY ELECTR (LEE) IMPACT DA Damage to pure dry samples of supercoiled DA is induced by bombardment with 3 ev electrons under a hydrocarbon-free 1 9 torr residual atmosphere (9). Plasmid DA [pgem 3Zf( ), 3199 base pairs] is first extracted from Escherichia coli DH5, purified, and resuspended in nanopure water into a clean -filled glove box, where the following manipulations are performed. An aliquot of this pure aqueous DA solution is deposited onto chemically clean tantalum substrates held at liquid nitrogen temperatures, lyophilised with a hydrocarbon-free sorption pump at.5 torr and transferred directly in a UHV chamber without exposure to air. After evacuation for 4 h, room-temperature DA solid films of 5 monolayer average thickness and 6 mm diameter are irradiated with a monochromatic LEE beam of the same diameter. Each target is bombarded for a specific time at a fixed beam current density (. 1 1 electrons.s 1.cm 1 ) and incident electron energy. After 4 h under UHV, the DA still contains Downloaded from at Main Library of Gazi University on May 4, 15 57

2 .5 H molecules per base pair as structural water (14). After bombardment, the DA is analysed by agarose gel electrophoresis and quantified as supercoiled (undamaged), nicked circle (SSB), full-length linear (DSB), and short linear forms (15). The measured yields reported in Figure 1 were found to be linear with electron exposure at each energy (9). The incident electron energy dependence of these yields (Figure 1) shows that LEEs induce SSBs and DSBs, even at electron energies below the ionisation limit of DA ( 8 ev) (16). Also, the damage is highly dependent on the initial kinetic energy of the incident electron, particularly below 14 to 15 ev, where thresholds near 3 to 5 ev and intense peaks near 1 ev are observed. The results shown in Figure 1 can be understood by investigating the fragmentation induced by LEEs to the various sub-units of the DA molecule, including its structural water. DA is composed of two strands of repeated sugar phosphate units hydrogenbonded together by the bases which are chemically linked to the sugar moiety of the backbone. A shortchain segment shown in Figure (b) exhibits the repeated sugar phosphate sub-units of the deoxyribose backbone to which are chemically bonded the bases DA breaks per incident electron (x1-4 ) DA loss per incident elctron (x1-4 ) DA double strand breaks (DSBs) DA single strand breaks (SSBs) Loss of super-coiled DA L. SACHE (a) (b) (c) adenine, cytosine and thymine. Results obtained from electron bombardment of solid films of DA constituents are presented in the following sections. These include H, the bases and the backbone sugar-like analogues, tetrahydrofuran (I), 3-hydroxytetrahydrofuran (II), and -tetrahydrofuryl alcohol (III) whose molecular structures are shown in Figure (a). LEE DAMAGE T THE DA-BACKBE SUB- UITS Figure 3 shows the H ion yields desorbed by the impact of 1 ev electrons on 1 ML films of I, II and III. The curves in Figure 3 are characterised by an onset at 6., 5.8, and 6. ev and a yield maximum centred at 1.4, 1., and 1. ev for I, II, and III, respectively. A second feature is also observed in the H yield function for II; it appears as shoulder on the low energy side of the 1 ev peak and is characterised by a sharp onset and a peak maximum near 7.3 ev. These results were generated by measuring with a mass spectrometer the H anions, desorbed by a collimated 4 na electron beam of 8 MeV full width at half maximum, incident at an angle of 7 from the surface normal of the sample film (17). The multilayer films of I, II and III were grown in UHV on an electrically isolated polycrystalline platinum ribbon attached to the tip of a closed-cycle helium-refrigerated cryostat (17). (a) (b) H H I II III P - P - H H Downloaded from at Main Library of Gazi University on May 4, Incident electron energy (ev) Figure 1. Measured yields, per incident electron, for the induction of DSBs (a), SSBs (b), and loss of the supercoiled DA form (c), in DA solids by electrons of 3 ev. The error bars correspond to one standard deviation of the measurement. Figure. Schematic drawing of (a) the DA backbone sugarlike analogues tetrahydrofuran (I), 3-hydroxytetrahydrofuran (II), and -tetrahydrofuryl alcohol (III), and (b) a short-chain segment of a single-stranded deoxyribose backbone of DA. 58

3 MECHAISMS F LW EERGY ELECTR DAMAGE T CDESED BIMLECULES AD DA All features below 15 ev in Figure 3 are characteristic of dissociative electron attachment (DEA) to I, II and III (i.e. the formation at a specific energy of a transient anion of the parent molecule which dissociates into H and the corresponding neutral radical). The steep rise in the H signal with an energetic threshold near 14.5 ev is characteristic of non-resonant dipolar dissociation (DD) of C H bonds in I, II, and III; it could also partially arise from DD of the H bond in II and III. DD results from the dissociation of an electronic excited state into a positive and a negative ion fragment. The formation of H via DEA from I, II and III has been discussed in detail by Antic et al (17). These authors considered the possibility of H arising from dissociation of the tetrahydrofuran ring, the H and the -CH H group. ther decay channels of the transient anions may result in the formation of larger anion fragments, such as the H,CH H, and the (M 1) ion fragment and compete with H production, but these heavier ions were not observed to desorb. Typically, large mass fragments do not possess sufficient kinetic energy to escape induced polarisation and thus remain trapped within the film (18). wing to the strong simi- H - ion yield (counts.s-1 x1 3 ) L I II III Electron energy (ev) Figure 3. H desorption yields stimulated by the impact of 1 ev electrons on a 1 monolayer (ML) thick film of I, II, and III. The small arrows indicate the positions of the two Feshbach resonances associated with dissociative electron attachment to the hydroxyl group in II and III, respectively. The smooth solid lines serve as guides to the eye, and the curve baselines have been shifted vertically for clarity. larity of the H desorption profiles for I, II and III, these authors concluded that the majority of the anion yield for all three systems arises from at least one transient anion associated with electron attachment to the furan ring and located near 1 ev. Considering the largely Rydberg character of the excited states in I near the energy range of the observed resonance, they further suggested that this resonant state is of the core-excited type, possibly with dissociative valence * configurational mixing. Lepage et al (19) have observed a broad resonance at similar electron energies in the excitation function of several vibrational modes in I, where they found good correlation with the gas-phase resonance observed in cyclopentane; they have tentatively assigned it as a core-excited shape resonance. This assignment is consistent with that made for the resonance observed near 1 ev for the (M 1) fragment (as well as all the lower mass fragments) produced by DEA to gaseous furan (). LEE DAMAGE T THE DA BASES Electron impact dissociation of DA bases has been investigated in the gas (1) and the condensed phase (1,11,13). In both phases, a large variety of stable anions is produced via DEA to the bases for electron energies below 3 ev. The anions H,,C,CH, C and CH were observed from gaseous thymine, whereas electron impact on gaseous cytosine resulted in the formation of the anions H, C, and/or H,C, C,C 4 H 5 3 and/or CH 4 3 (11) and C 3 H 3. All four bases were investigated in the form of thin multilayer films held at room temperature, but fewer anions of different masses were measured than in the gas phase. The difference is principally due to the inability of the heavier anions to overcome the polarisation potential they induce in the film, causing them to remain undetected in the target (18). In fact, only the light anions H,,H,C, C and CH were found to desorb by the impact of 5 3 ev electrons on the physisorbed DA bases adenine, thymine, guanine and cytosine via either single or complex multibond dissociation (11). As an example, the C yield functions produced by 3 ev electron impact on thin films of each of the four bases are shown in Figure 4. All yield functions exhibit maxima which are characteristic of DEA to the bases. Whereas anions such as H can be induced via a simple bond cleavage from the bases, the production of C occurs under more complex ring dissociation. From the structure of the DA bases, it can be readily seen that extraction of a C anion must involve more than a single bond cleavage. In other words, during the lifetime of the negative ion, not only exocyclic single bond occurs, but also complex endocyclic multibond cleavages are involved via most likely either stepwise () or concerted reactions (3). The similarity of the C,H and ion yield functions with resonant peaks at 9. ev and 1 ev from guanine Downloaded from at Main Library of Gazi University on May 4, 15 59

4 or cytosine and thymine films, respectively, observed by Abdoul-Carime et al (11), suggests that they arise from the formation of a same excited isocyanic anion intermediate (CH)*, via closely lying but distinct resonances; i.e. (G,C,orT ) R + (CH)*, where R represents the remaining radical; then (CH)* further undergoes fragmentation into different possible dissociative channels: C + H, H + C or + CH. L. SACHE LEE DAMAGE T AMRPHUS ICE FILMS Previous results from electron-bombarded amorphous H and D films are also of relevance to DA damage, since on average.5 H molecules were estimated to be present for each base pair in the DA samples used (9) to produce the data shown in Figure 1. The damage induced by low energy electron impact on amorphous ice films has been measured by recording C - ion yields (counts.s -1 ) (a) Adenine (b) (c) Guanine (d) Downloaded from at Main Library of Gazi University on May 4, Thymine Cytosine Incident electron energy (ev) Figure 4. Incident electron energy dependence of C ion yields desorbed under 5 na electron bombardment of 8 ML thick films of (a) adenine, (b) thymine, (c) guanine, and (d) cytosine. 6

5 MECHAISMS F LW EERGY ELECTR DAMAGE T CDESED BIMLECULES AD DA H (4),H (5,6),D( S), ( 3 P) and ( 1 D ) (7) desorption yield functions in the range 5 3 ev. Most of these functions exhibit resonance structures below 15 ev which are characteristic of transient anion formation. From anion yields, DEA to condensed H was shown to result principally into the formation of H and the H radical from dissociation of the B 1 state of H located around 8 ev with smaller contributions from the A 1 and B anionic states formed near 9 and 11 ev, respectively (4). At higher energies non-resonance processes such as dipolar dissociation, lead to H fragmentation with the assistance of a broad resonance extending from to 3 ev (4,6). DISCUSSI F THE DA RESULTS In previous sections, it has been shown that the incident electron energy dependence of the damage to elementary constituents of DA, probed in the form of desorbed anions, exhibits strong variations due to DEA. From comparison of these maxima in the anion desorption yield functions of these constituents, to the DA results, it becomes quite obvious that the strong energy dependence of the DA strand breaks below 15 ev in Figure 1 can be attributed to the initial formation of transient anions, decaying into the DEA and/or dissociative electronic excitation channels. However, since the basic DA components (i.e. the sugar and base units and structural H ) can all be fragmented via DEA between 5 and 13 ev, it is not possible to attribute unambiguously SSBs and DSBs to the initial dissociation of a specific component. For example, the maximum in the SSB yield lying near 8 ev is very close to the maximum in H + H production from H, but the DA bases also produce H and the corresponding radical with high efficiency near 9 ev (11). What appears to be more convincing is the coincidence in the 1 ev peak in H production in Figure 3 from tetrahydrofuran and its analogues, with that in the DSB yield seen in Figure 1. This is not surprising, since two events are necessary to create a DSB, and the probability for such breaks is likely to be larger when the first hit results in a SSB (i.e. in a break on the sugar phosphate backbone). Another interesting aspect of the DSB yield curve is the absence of damage induced by direct (non-resonant) electronic transitions. Indeed, the two points in Figure 1 around 14 and 15 ev lie at the zero base line, indicat- ing that below 16 ev a DSB occurs exclusively via the decay of transient anions. These observations suggest that below 16 ev, a DSB occurs via molecular dissociation on one strand initiated by the decay of a transient anion, followed by reaction of at least one of the fragmentation products on the opposite strand. This hypothesis is further supported by the observation of electron-initiated fragment reactions (such as hydrogen abstraction, dissociative charge transfer, atom and functional group exchange, and reactive scattering) occurring over distances comparable to the DA s doublestrand diameter ( nm) in condensed films containing water or small linear and cyclic hydrocarbons (8 3). SUMMARY Results on the damage induced by low energy electrons to DA and some of its basic constituents have been reviewed in this article. The incident electron energy dependence of the yield of anions desorbed from solid H, sugar phosphate backbone sub-units and DA bases exhibits strong variations typical of dissociative electron attachment to these molecules. These results were compared to the measured yields per incident electron for the induction of SSBs and DSBs of supercoiled DA measured between 3 and ev. Such a comparison shows that below 15 ev, the DA damage results principally from the formation of transient molecular anions localised on the DA s basic components. These transitory states arise from the fundamental electrodynamic and exchange forces acting between the electron and a specific sub-unit of the DA. Because of the universality of these fundamental interactions, they are expected to also be operative in living cells. Thus, from a radiobiological perspective, the results summarised in this article suggest that the abundant low energy ( ev) secondary electrons, and possibly their ionic and radical reaction products, play a crucial role in the nascent stages of cellular DA radiolysis and may already induce substantial damage long before their thermalisation along ionising radiation tracks. ACKWLEDGEMETS The author would like to thank Ms Francine Lussier for the preparation of this manuscript. This work was supported by the Canadian Institutes of Health Research. 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