and Double-Strand Breaks of Dry DNA Exposed to Protons at Bragg

Jan 3, 2017 - dose−depth distributions, especially due to the significant increase of the dose profile at the end of the particle range: the so-call...
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Article

Single and Double Strand Breaks of Dry DNA Exposed to Protons at the Bragg-Peak Energies Mounir Souici, Talat Tariq Khalil, Dominique Muller, Quentin Raffy, Rémi Barillon, Abdelfettah Belafrites, Christophe Champion, and Michel Fromm J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b11060 • Publication Date (Web): 03 Jan 2017 Downloaded from http://pubs.acs.org on January 7, 2017

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The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Single and double strand breaks of dry DNA exposed to protons at the Bragg-peak energies Mounir Souici 1,2, Talat T. Khalil1, Dominique Muller3, Quentin Raffy4, Rémi Barillon4, Abdelfettah Belafrites2, Christophe Champion5 and Michel Fromm1* 1

Université de Bourgogne Franche-Comté, UMR CNRS 6249 Chrono-Environnement, 16 Route de Gray, 25030 Besançon Cedex, France.

2

Laboratoire de Physique des Rayonnements et Applications, Université de Jijel, B.P. 98 Ouled Aissa, Jijel 18000, Algérie.

3

Laboratoire ICube, CNRS-Université de Strasbourg, 23 rue du Loess, 67037 Strasbourg, France 4

Institut Pluridisciplinaire Hubert Curien, UMR CNRS 7178, 23 rue du Loess, BP 28, 67037 Strasbourg Cedex 2, France.

5

Université de Bordeaux, CNRS/IN2P3, Centre d’Etudes Nucléaires de Bordeaux Gradignan, CENBG, Chemin du Solarium, BP120, 33175 Gradignan, France.

Corresponding Author Michel Fromm, [email protected], Tel: (0) 33 381 666 560, Fax: (0) 33 381 666 522

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ABSTRACT Ultra-thin layers ( 0.99) whose slopes provide a direct measurement of the SSB yields expressed in units of [plasmid-1 . Gray-1]. These yields were found to be equal to 10-4 SSB.plasmid-1.Gy-1, for both energies of 3000 and 1500 keV. In the energy range 500 keV - 90 keV, more complex functions were needed to accurately fit the experimental data.

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4

0,06

3000 keV 1500 keV 500 keV 185 keV 155 keV 140 keV 90 keV

0,05 0,04

-1 SSB (plasmid )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3

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0,03 0,02 0,01 0,00

2

0

200

400

600

800 1000

1

0 0,0

5,0e+4

1,0e+5

1,5e+5

2,0e+5

2,5e+5

Dose (Gy)

Figure 4. SSBs per plasmid (see Table 2) plotted as a function of the absorbed dose at various proton energies. The dashed lines are mathematical fits of the experimental data (see text for more details). As average values stem from three measurements, error bars are fixed at the highest found SD values (Table 2) in order to increase the determination of uncertainty; namely 10% of the average yield. Error bars for doses are those recorded in Table 2 and stem from uncertainty propagation in determining dose values (relations 3 and 4).The inset shows the tangents through the origin for each curve (see also text for more details) whose slope provide a direct quantification of the damage rates expressed in SSB . Gy-1 . bp-1 or in SSB . Gy-1 . Da-1 (Table 2).

Thus, data for 500 keV protons were best fitted by an exponential-growth model f1=a*(1-exp(b*x)) (R2 = 0.9969) that provided - by first derivative at the “zero dose” - a yield of 1.1×10-4 SSB.plasmid-1.Gy-1. As for the proton energies 185, 155 and 140 keV, best fits (R2= 0.9819; 0.9778 and 0.9999, respectively) were obtained by using 4 parameters exponential functions f2=a*(1-exp(-b*x))+c*(1-exp(-d*x)). Corresponding DNA SSB yields were finally calculated thanks to the “zero dose” derivatives: 1.9×10-4, 1.2×10-4 and 10-4 SSB.plasmid-1.Gy-1, for 185, 155 and 140 keV, respectively. As for energy 90 keV, a yield of 8×10-5 SSB.plasmid-1.Gy-1 was obtained by using the above-cited f1 function. However, the latter result appears somewhat as a

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matter of concern due to the poor coefficient of goodness of the fit (R2 = 0,6668). Finally, the yields expressed in SSB.Gy-1.bp-1 were calculated by using the pBR322 total number of base pairs (4363 bp, 2.859×106 Da). Similarly to the previous SSB quantification, Figure 5 reports on the experimental DSBs yields per plasmid as a function of the absorbed dose as well as an illustration of the damage rate quantification in DSB . Gy-1 . bp-1 (see inset of Figure 5) or in DSB . Gy-1 . Da-1 (see Table 3). Energies (keV)

LET R2 (keV.µm 1 )

n(SSB) x n(SSB) R2 -8 -11 10 x 10 -1 -1 Gy . bp Gy-1. Da1

n(DSB) n(DSB) SSB/DSB x 10-9 x 10-12 Gy-1. bp- Gy-1. Da1

1

3000

15

0.9938

2.29

3.50

0.9904

0.63

0.96

36

1500

25

0.9986

2.29

3.50

0.9936

0.73

1.11

31

500

50

0.9969

2.52

3.85

0.9965

1.32

2.02

19

185

89

0.9819

4.35

6.65

0.9885

3.28

5.00

13

155

95

0.9778

2.75

4.20

0.8978

2.87

4.37

10

140

99

0.9999

2.29

3.50

0.8668

1.42

2.16

11

90

110

0.6668

1.83

2.80

0.5105

1.72

2.63

7

Table 3. Experimental SSB and DSB yields expressed in breaks.Gy-1.bp-1 and in breaks.Gy-1.Da-1 for the various proton energies investigated here. Goodness of fits also is reported (see insets in Figures 4 and 5). Underlined numbers are data that obviously are distorted by the false quantification of the F fraction, which is confirmed with rather wrong values of the coefficients of determination. The ratios SSB/DSB are also presented.

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0 , 1 3000 keV 1500 keV 500 keV 185 keV 155 keV 140 keV 90 keV

-1

DSB (plasmid )

8 , 0 6 , 0

0,08

4 , 0 0,06 0,04

2 , 0

0,02 0,00

0 , 0

0

2000

4000

6000

8000

5 + e 3

5 + e 2

5 + e 1

0

Dose (Gy)

Figure 5. Same as in Figure 4 for DSB quantification. Error bars are fixed at the highest found SD values (Table 2), namely 25% of the average yield. Finally, Figure 6 reports the SSB/DSB ratio, which exhibits a reasonable linear character (R2 = 0.9600) when plotted as a function of the proton LET (see discussion for more details). 50

SSB/DSB = 37.8394 - 0.2855 LET 2 (R = 0.9600)

40

SSB/DSB

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30 20 10 0 0

20

40

60

80

100

120

-1

LET (keV.µm )

Figure 6. SSB/DSB ratio as a function of the proton LET (linear regression appears as dashed line).Error bars stand for the accumulation of errors in determining SSB and DSB yields (see text and legends of Figures 4 &5). Cross-sections

Single strand breaks The transformation of the topological form of a DNA plasmid from the supercoiled to the relaxed (i.e. circular) form is the result of a single strand break.28 As originally proposed by Katz ,32 in the present case, the corresponding fraction of supercoiled plasmid DNA may be written as

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N N 0 = exp ( −σSSB φ )

(5)

where N and N0 stand respectively for the % of supercoiled topology in the sample after irradiation at a fluence φ and the initial % of supercoiled form. In this context, σSSB refers to the cross-section for a single strand break induction. Thus, to extract the σSSB values we first analyze the data related to 3000, 1500 and 500 keV. For those energies, no fragmented DNA is observed provided that the fluence ≤ 1012 cm-2. A simple slope extraction is thus performed by using a linear fit (see Figure 7) that provides the following SSB cross-sections: 1.91×10-12, 3.55×10-12 and 4.46×10-12 cm2 for 3000, 1500 and 500 keV proton energies, respectively. As for energies 185, 155, 140 and 90 keV more important amounts of fragmented DNA appear at the higher fluences, the tangents to the curves taken at the “zero dose” have then been used to determine the cross-sections. This was done using the best fitting-functions, namely, f1= a1*{1exp(-b1*x)}+c1*{1-exp(-d1*x)} for 185 and 155 keV and f2=y2+a2*exp(-b2*x) for 140 and 90 keV (se Figure 7). The corresponding cross sections (σSSB) are reported in Table 4. 0

185 keV 155 keV 140 keV 90 keV

0,0

-1

Ln (N/N0)

-0,5

Ln (N/N0)

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-2 Ln (N/N0) = - 1.91 10-12 φ, R2=0.99

-3

Ln (N/N0) = - 3.55 10-12 φ, R2=0.99

-1,0

-1,5

Ln (N/N0) = - 4.46 10-12 φ, R2=0.98

-4

3000 keV 1500 keV 500 keV

-5 0

2e+11

-2,0

4e+11

6e+11

8e+11

1e+12 -2

Proton fluence, φ (cm )

0

2e+11

4e+11

6e+11

8e+11

1e+12

-2

Proton fluence, φ (cm )

Figure 7. The left side plot depicts the evolution of the logarithmic fraction of supercoiled pBR322 plasmids as a function of proton fluence for 3000, 1500 and 500 keV. The dashed lines are linear regressions; all have R2 ≥ 0.98. The plot on the right side concerns protons with energies 185, 155, 140 and 90 keV. The used fitting functions (dashed lines) are described in the text, all have R2 = 0.99; therefore no error bars are not presented.

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Double strand breaks Similarly, the DSB cross-sections are also determined by using the DSBs data plotted as a function of the fluence. In that case indeed, the cross-sections can be considered as being the reciprocals of the protons fluence giving one double strand break.33 Thus, as stated above for the SSB quantification, we still use linear functions for 3000 and 1500 keV protons whereas we use the first derivative at the “zero dose” of the best fits (namely f=a*(1-exp(-b*x)) for 500, 185, 155, 140 and 90 keV (see Figure 8). The corresponding σDSB cross sections are reported in Table 4. 3000 keV 1500 keV 500 keV 185 keV 155 keV 140 keV 90 keV

1,0

DSB (plasmid-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0,8

0,6

0,4

0,2

0,0 0

1e+12

2e+12

3e+12

4e+12

5e+12 -2

6e+12

Proton fluence (cm )

Figure 8. Evolution of the DSBs per plasmid as a function of the fluence for various proton energies (dashed lines, fit of the data, R2 > 0.99 except for 3000 keV, R2 = 0,9837). Error bars see Figure 5.

15

σSSB × 10-11 Standard deviation σDSB × 10-13 (cm2) of σSSB (x 10-11 (cm2) cm2) 0.19 0.09 0.39

Standard deviation of σDSB (x × 10-13 cm2) 0.10

1500

25

0.36

0.09

0.61

0.15

500

50

0.45

0.30

5.69

1.42

185

89

2.15

0.46

14.54

3.64

155

95

1.43

0.26

16.57

4.14

140

99

1.42

0.20

7.16

1.79

90

110

0.60

0.23

6.86

1.72

Energy (keV)

LET (keV.µm-1)

3000

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Table 4. Experimental cross-sections for SSBs and DSBs for various proton energies belonging to the Bragg-peak (90-185 keV) and to the Bethe-Bloch regions (500-3000 keV). The σSSB standard deviations result from the regressions in determining the tangent at dose zero, as for σDSB error bars they have been fixed to 25% of the respective cross-sections (see legend of Figure 5).

G-values for single strand breaks Radiochemical yields or G-values may be determined by using the following relation:30

G=

σN L

(6)

where N refers to the number of targets per unit volume (0.49 mol.m-3) and L is the LET. Eq (6) is in particular commonly used for scintillation counters or thermoluminescent dosimeters, considered as “one hit” detectors. In a first approximation, Eq (6) could thus provide G(SSB) values. In our experiments, we can consider that N is constant because the volume of the drop deposited on the Mylar foil is constant and the DNA plasmid concentration is constant too. LET values are presented in Table 4. Additionally, G-values can also be determined - at least for the SSB induction - by using the D37.20,34 Indeed, assuming Poisson statistics for SSB induction, the D37 value represents the radiation dose required to give on average one SSB per plasmid molecule. Thus, from the data reported in Figure 4 and Table 3 we can extract the D37 for each of the proton energies used in this study. In that case, G-values are finally deduced by using the following relation: G(SSB) = (1000/mwt)/D37

(7)

where mwt is the molecular weight of pBR322 DNA plasmid (2.859 MDa).

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G(SSB) ×10-7 (6) mol.J-1 3000 0.39 (±0.04) 1500 0.44 (±0.05) 500 0.28 (±0.03) 185 0.74 (±0.08) 155 0.46 (±0.05) 140 0.44 (±0.05) 90 0.17 (±0.02) Table 5. G-values for a SSB at various proton Energy (keV)

D37 ×103 Gy 10.00 10.00 9.09 5.26 8.33 10.00 12.50 energies. The D37

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G(SSB) ×10-7 (7) mol.J-1 0.35 (± 0,04) 0.35 (± 0,04) 0.39 (± 0,04) 0.67 (± 0,07) 0.42 (± 0,04) 0.35 (± 0,04) 0.28 (± 0,03) values used to compute the

radiochemical yields (see Eq (7)) registered in the last column are also reported. G-values are accompanied with their respective uncertainties (between parentheses) determined based on propagation of errors.

DISCUSSION The yields for SSB and DSB per Gray and per Dalton determined herein are in fairly good agreement with the available comparable data, in particular with the data reported by Ushigome

et al.20 In that particular study, alpha particle irradiations of fully hydrated films of pUC18 DNA plasmid were performed at 5.68 °C leading to SSB and DSB yields of same order of magnitude than ours at comparable LET (see Table 6). However, it has been shown35 that damage yields depend also on the projectile atomic number for iso-LET ions; with SSB yields increasing with the atomic number while DSB yields decrease at constant LET. Such a trend is observed for SSB yields while it is not the case for DSB yields (Table 6). For these reasons and in order to remain cautious, we have limited our comparison to helium ions for which the difference in atomic number with proton is the lowest.

Alpha Particle LET

SSB yield DSB yield Proton SSB yield DSB yield (Gy-1.Da-1) (Gy-1.Da-1) LET (Gy-1.Da-1) (Gy-1.Da-1) [20] [20] (keV.µm-1)

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(keV.µm-1) 19 6.9×10-11 0.96×10-12 15 3.5×10-11 0.96×10-12 -11 -12 -11 95 6.7×10 10.9×10 95 4.2×10 4.4×10-12 Table 6. Comparison between our current proton SSB and DSB yields and the alpha particle homologous taken at comparable LET.20

Besides, it is worthy to mention that determinations of single and double strand break yields in such fully hydrated plasmid DNA films20 have shown that the ratios of {prompt /(prompt + heatlabile damages)} participating to nSSB and nDSB remain extremely close to 1 for 2.2 ≤ LET ≤ 95 keV.µm-1. We can thus consider that within our experimental framework, heat-labile damages might not contribute substantially to the measured yields. Similarly, in a different experimental approach, Prise and coworkers36 exposed 2.65 ml aliquots of plasmid (pMSG-CAT) solution placed under a glass coverslip on a mylar based dish (3 µm thick) - to 3.5 MeV α-particles. Let us note that in such conditions, the LET at the entry of the sample is 110 keV.µm-1 and thus should cover the Bragg-peak region when α−particles are slowed down in the sample. Interestingly, the authors used different scavenging conditions and notably high scavenger concentrations (namely 200 mM Tris); that allow considering the indirect effects as negligible. The yields provided by these experiments exhibit a quite satisfactory agreement with our data (see Table 3) at same initial LET, namely, 7.6×10-8 SSB.Gy-1.bp-1 (4.2 time the yield we measured) and 1.5×10–8 DSB.Gy-1.bp-1 (8.7 time the yield we measured). More recently, experimental data for SSB and DSB yields in dry DNA plasmid (pBR322) samples exposed to 10, 20 and 30 MeV protons (LET = 6.39, 3.64 and 2.61 keV.µm-1, respectively) have also been published.21 The authors find SSB yields of (89.0 ± 3.4)×10-9 and (92.9 ± 7.1)×10-9 Gy-1.bp-1 by using Cowan and McMahon models, respectively, for 10 MeV protons (LET = 6.39 keV.µm-1) whereas we find 22.9×10-9 SSB.Gy-1.bp-1 for 3 MeV protons

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(LET = 15 keV.µm-1). Regarding the DSB yields, the authors report the value of (3.0 ± 0.5)×10-9 DSB.Gy-1.bp-1 with the Cowan model and that of (2.8 ± 0.5)×10-9 DSB.Gy-1.bp-1 when the McMahon model is used. Comparatively, we find 0.63 10-9 SSB.Gy-1.bp-1 for 3 MeV protons (LET = 15 keV.µm-1). In the same way, the data provided by Fulford and coworkers37 who developed an experimental device allowing to expose aqueous solutions of pUC18 plasmid DNA maintained at 277 K to 3.2 MeV α-particle (LET in water = 120 keV.µm-1) are in satisfactory agreement with ours. Actually, the SSB yield they determined experimentally at high scavenging capacity (typically mimicking the cellular environment) is nSSB = 7.4 10-11 Gy-1.Da-1 and we find for LET in DNA = 110 keV.µm-1, nSSB = 2.8 10-11 Gy-1.Da-1. DSB yields are in fact not fairly comparable because at this LET value, (see Table 3) the goodness of fit was of poor quality. Leloup et al.,38 published SSB and DSB yields for plasmid DNA (pHAZE) irradiated with 1.03, 19.3 and 249 MeV protons (LET of 25.5, 2.7, and 0.39 keV µm–1 respectively) in aqueous phase and in the presence of 200 mM glycerol (a hydroxyl radical scavenger with equivalent scavenging capacity than Tris). With 1.03 MeV protons (LET = 25.5 keV µm–1), the authors find the following yields: nSSB ≈ 7×10-10 Gy-1.Da-1 and nDSB ≈ 2×10-11 Gy-1.Da-1 to be compared to our measurements, namely, nSSB ≈ 3.5 10-11 Gy-1.Da-1 and nDSB ≈ 1.11 10-12 Gy-1.Da-1 corresponding to LET = 25 keV µm–1. There is at least a one order of magnitude variation, our data being systematically lower. In this particular case, it may be likely that the radicals produced on the glycerol molecules further to hydroxyl radicals scavenging, at their turn attack the plasmids as well. It was actually shown recently that side-effects of that type arise when arginine is used as an •OH scavenger.39 We also observe a lower agreement with the data presented by others40 stemming from proton track simulations that were carried out in small micrometric volumes

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representing small DNA containments using an adapted DBSAN algorithm. In these studies, proton energies ranged from 500 keV to 50 MeV and SSB and DSB yields at 500 keV are typically 10-fold higher than ours. Based on a numerical simulation which accounts for five organization levels of the human genetic material, namely, the nucleotide pairs, the double helix, the nucleosomes, and the 10 nm and 30 nm chromatin fibers but also the three possible double helical structures A, B and Z of DNA, Bernal and coworkers41 also provide a yield of nSSB = 120 DBS.Gy-1.Gbp-1 for protons with LET= 60 keV.µm-1 slowed down in B DNA. Comparatively, we find here 25.2 SSB.Gy-1.Gbp-1 for LET= 50 keV.µm-1 (500 keV protons) with pBR322 DNA in its native B form, i.e. without proteins wrapping DNA. Similarly, these authors provide nDSB = 22 DBS.Gy-1.Gbp-1 (still for a proton of LET= 60 keV.µm-1) while we here report nDSB = 1.32 DBS.Gy-1.Gbp-1 for 500 keV protons (LET= 50 keV.µm-1). Under these conditions, the numerical yields reported therein appear as systematically higher than ours. However, it is worth noting that in their study, the authors didn’t account for complex DSBs whose role may be crucial in the DSB quantification and only report on “strand break yields (which may ) … be analyzed from a relative point of view”.41 Besides, Figure 8 clearly shows that DNA fragmentation becomes important starting from 5×1011 protons.cm-2 at the 500 keV proton energy (i.e. at the same proton energy used in reference 41), that may explain why our yields are systematically lower than the existing computed values (provided by numerical simulations that do not account for the fragmentation process) and would then shade some light on the way in which DNA is damaged. Thus, the deviation from linearity reported for the SSB and DSB yields versus fluence in Figs. 4 and 8 and notably the fact that SSB and DSB yields plateau-out over a given fluence, can be interpreted by taking into account the radial dose distribution of secondary electrons. Indeed,

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severe DNA fragmentation in plasmids may require multiple localized events in the close vicinity of few consecutive base pairs of the DNA macromolecule. Supralinearity and saturation in the response of thermoluminescent (TL) materials as a function of heavy charged particle dose was theorized by Horowitz and coworkers42 including nearest-neighbor track interactions. An increase of track overlap probability at high doses (fluences) may be interpreted phenomenologically just as an increase in the hit probability on a same target. The occupation probabilities as a function of dose (or fluence) can usually be described using a linear/exponentially saturating function:42 n Break = N max Break [1 − exp( −Sφ) ]

(8)

The occupation probability, F(φ), is then defined as the fraction of the number of damages (SSB or DSB); nBreak per plasmid inflicted by the particle at a given fluence φ to the maximum yield (i.e. at saturation of the measured yield); N max Break . In this context, S should hence refer to the average area of the radial extension of a proton track in the plane of the sample. This concept allows implicitly to define rmax= S π as the distance from the proton track axis up to which the occupation probability for a given damage (SSB or DSB) would tend to 100%. Mathematical fits realized on data by using functions of the type of Eq (8) may therefore provide information on the radial dimensions of the proton tracks in the ultra-thin DNA layers used in the present study. Over the seven data sets of DSB versus fluence (Figure 8), functions of the type of Eq (8) were used for 5 of them, as for SSB, only one data set, namely for 500 keV, exhibits a behavior in line with Eq (8). All mathematical fits having R2 > 0.99, we present in Figure 9 the rmax values relative to DSB as a function of the respective proton energies. The rmax value for a SSB at 500 keV is 4.7 nm (rmax = 5.2 nm for DSB at the same energy). Figure 9 confirms the sharp increase of damaging probability as proton energy decreases down to the

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Bragg-peak region. Besides, the fraction of a proton energy loss deposited within a 10 nm track radius area was determined by others for various energies in water.43 Radial extension equivalent to 100% DSB occupation probability

12

10

rmax (nm)

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8

6 rmax (nm)=5.1821+25.2583*exp(-0.0152*E(keV))

4

R2 = 0.9892 0

100

200

300

400

500

600

Proton energy (keV)

Figure 9. Radial extensions of proton tracks in dry DNA at various energies for which the occupation probability is 100% (see text for additional details). Error bars stand for propagation of uncertainty including the fitting procedure (Eq. 8). This fraction appears as being greater than 90 % for 500 keV protons and increases sharply when the proton energy decreases below 500 keV. In the present study, we use a DNA with density = 1.4 g.cm-3 and thus the stopping power of the DNA samples must be higher than for liquid water. Aiming at demonstrating that the choice of the source size and type could modify the dose distribution and then influence the DNA damage rates, Pater and coworkers44 have recently demonstrated that in the case of 1.5 keV proton simulations, the radial dose distribution within a 15 nm region of interest (ROI) strongly depends on the type of proton beam used. Very interestingly, they showed that the radial dose distribution in the 15 nm diameter ROI is extremely sensible to the type of beam. Thus, when a pencil beam is used instead of a plane beam, the radial dose distribution is much higher near to the ion’s path and then decreases exponentially up to distances of 6-8 nm where it finally stabilizes. In this context, our estimated

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radial extensions of proton tracks in the Bragg-peak region are in very good agreement with calculated ones. This reinforces the soundness of the present approach and allows a better understanding of the way by which fragmentation occurs when solely the direct effects are taken into account. We provide thus experimental evidence that DNA fragmentation due to the sole direct effects is mainly a consequence of proton track overlapping, or in other words, that fragmentation happens in the intersection areas of neighboring proton tracks due to cumulative events. Also relevant to those who are developing computer codes for the prediction of DNA damages by direct effect, we have shown that within the studied energy range of protons, when the ratios of y= SSB/DSB are plotted as a function of the corresponding LETs, an affine relation is found: y = 37.84 - 0.29 LET (keV.µm-1). Such a linear behavior of the direct effects versus LET was also suggested by Dos Santos and co-workers45 who assessed the influence of the chromatin density on the number of clustered damages created by protons for different types of cell nuclei with the GEANT4 simulation tool. Besides, experimental SSB and DSB cross sections have been reported in Table 4, those latter are represented as a function of proton LETs in Figure 10. The shapes of the curves are typical of the well-known track structure models developed to simulating strand breaks in DNA (or in cells) after heavy ion irradiation, they exhibit in particular a well-documented hook observed at the high LET values.11,46,47 To the best of our knowledge, experimental cross-section data for dry DNA precisely stemming from proton irradiations in the Bragg-peak energy region were not available up to now. Finally, the G-values for a single strand break, G(SSB), in dry DNA at the proton energies of the Bragg-peak region have also been determined in this study (Table 4). The highest SSB G-value arises for proton energy of 185 keV, for which almost all measured yields and cross-sections peak; only the DSB cross-section is maximum at 155 keV. Significantly, Ushigome and coworkers,20 find exactly the

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same G(SSB)= 0.67 10-7 mol.J-1 with alpha particles of the same LET as 185 keV protons (95 keV.µm-1). Taking into account the data presented herein, one can conclude that highest damage rates in DNA occur for proton energies in the range 150 – 200 keV.

2

σSSB, σDSB (µm )

1e-3

1e-4

1e-5 σ

B S S

σ

B S D

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1e-6 10

-1

100

LET (keV.µm )

Figure 10. Experimental cross-sections for SSB and DSB induction in pBR322 plasmid DNA as a function of proton LET. Dashed lines are drawn to guide the eyes. Error bars see Table 4.

CONCLUSION Yields for DNA topological modifications as well as cross-sections for SSB and DSB have been measured along with G(SSB) values for dry and ultra-thin plasmid DNA layers exposed to protons in the Bragg-peak energy region. Fragmentation of the DNA plasmids is found to be important, especially for the lower particles energies (higher LETs) but also for proton fluences greater than 1×1011 cm-2. Analysis of the data gathered herein suggests that fragmentation is mainly the result of cumulative events arising due to proton track overlapping when the sole direct effects are taken into account. Besides, in addition to SSB and DSB, we pointed out a regime of DNA destruction reached for proton fluences higher than 1×1011 cm-2. In this particular regime, the fragmentation process is so strong that the AGE measurements exhibit long smears, which are in some cases no more

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detected by the analysis tool i.e. with resulting DNA fragments extremely small revealing then a desirously damaged DNA double helix. AGE analyses as used in the current study cannot provide fragment proportions comparable to SSB or DSB abundances. More importantly, we also observed the destructive regime of high fragmentation at proton fluences lower than 1×1011 cm-2 provided that the proton energy is low enough, namely near to the end of the Bragg-peak energy region (< 200 keV). In this particular region, we observe anticipated hooks for both SSB and DSB yields as well as cross-sections when plotted versus LET. All the physical quantities determined in this study, except rmax, attain their highest values near to, but not at the Bragg-peak, namely in an energy range of 150 – 200 keV. Based on an occupation probability model, we have also shown that the radial extension of damaging efficacy expressed in terms of DSB increases as and when the proton energy decreases. This is in line with the explanation of the observed increase of fragmentation, which in consequence, might be due to the riddling of DNA components by protons and their secondaries. Such a point of view is not dissimilar to the well-known “saturation” or “overkill” effects of track structure theory based on radial dose distributions (hit-theory). Lastly, let us note that the present experimental data may be of particular interest for all those who are developing computer simulation tools, SSB and DSB cross-sections and yields being of prime importance for numerical developers as well as researchers aiming at refining the proton therapy treatment planning. In a near future, the scheduled work plan will consist in exposing layers in which plasmid DNA is complexed to polypeptides in order to mimic its interaction with histone proteins.

ACKNOWLEDGEMENTS: We would like to thank the Programme National Exceptionnel; PNE (French-Algerian Collaboration program) for the financial grant allocated to Dr. Souici in order to work during 18 months in our laboratory in France. Part of this work also was supported by a grant from the Iraqi embassy (Ph. D. of T. T. Khalil).

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