Strong Strand Breaks in DNA Induced by Thermal Energy Particles

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A: Kinetics, Dynamics, Photochemistry, and Excited States

Strong Strand Breaks in DNA Induced by Thermal Energy Particles, and Their Electrostatic Inhibition by Na+ Nanostructures Upendra Nayek, Vayakkara Kolaprath Unnikrishnan, Abdul Ajees Abdul Salam, Parinda Vasa, Santhosh Chidangil, and Deepak Mathur J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.9b00650 • Publication Date (Web): 28 Mar 2019 Downloaded from http://pubs.acs.org on March 29, 2019

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The Journal of Physical Chemistry

Strong Strand Breaks in DNA Induced by Thermal Energy Particles, and Their Electrostatic Inhibition by Na+ Nanostructures Upendra Nayek,a,b V. K. Unnikrishnan,a,c Abdul Ajees Abdul Salam,a,b Parinda Vasa,d Santhosh Chidangil,a,c and Deepak Mathura,*

a

Department of Atomic and Molecular Physics, Manipal Academy of Higher Education,

Manipal 576 104, India b

Centre for Applied Nanosciences, Department of Atomic and Molecular Physics, Manipal

Academy of Higher Education, Manipal 576 104, India c

Centre for Biophotonics, Department of Atomic and Molecular Physics, Manipal Academy of

Higher Education, Manipal 576 104, India d Department

of Physics, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India

ACS Paragon Plus Environment

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ABSTRACT Low-power laser pulses of 6 ns duration (1064 nm wavelength) have been used to create plasma in an aqueous solution of plasmid DNA (pUC19). Thermal-energy electrons and .OH-radicals in the plasma induce strand breakages in DNA, including double strand breaks (DSBs) and possible base oxidation/base degradation. The time-evolution of these modifications shows that it takes barely 30 s for damage to DNA to occur. Addition of physiologically relevant concentrations of a salt (NaCl) significantly inhibits such damage. We rationalize such inhibition using simple electrostatic considerations. The observation that DNA damage is induced by plasma-induced photolysis of water suggests implications beyond studies of DNA, and opens new vistas for using simple nanosecond lasers to probe how ultra-low energy radiation may affect living matter under physiological conditions.


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Introduction DNA is a very stable, naturally-occurring, long-chain polymer, with a half-life toward spontaneous hydrolysis of ~130,000 years.1 Nature requires that, concomitantly with mechanical robustness, DNA must also be able to sustain large conformational changes - bending, compression, twisting2 - in order to effectively pack into chromosomes and to perform tasks like replication and transcription. However, it is well known that energetic particles induce damage to DNA by breaking one or more of strands or by inducing oxidation of bases (guanine).3,4 Such damage, which can lead to cellular transformations, may be induced naturally from either terrestrial or extra-terrestrial sources. Extra-terrestrial sources include the pervasive cosmic rays while X-rays, γ-rays, and high-energy electrons are the most common energetic terrestrial sources. In the case of human cells, several thousand damage events, like strand breakages, can occur per cell per day. Estimates for rates of oxidative damage vary from a few thousand5,6 to about 10,000 per cell per day;7,8 up to 55,000 single stand breaks may occur per cell per day in mammalian cells,9 with the corresponding number of double strand breaks being in the range of a few tens per cell per day.10,11 However, nature has ensured that internal repair mechanisms come into play such that the rate of damage in a healthy cell is more or less balanced by the rate of repair. Frequencies with which naturally-occurring DNA damages arise per day as a consequence of endogenous cellular processes have been compiled.12 In a diseased cell, however, the rate of repair is less than the rate of damage, and this can lead to consequences like senescence (an irreversible state of dormancy), apoptosis (amounting to cell suicide), or unregulated cell division (leading to formation of a tumour that may be cancerous). DNA damage may take one of two forms: (a) either only a single strand breaks (SSB), in which case the complementary strand is able to act as a template to biochemically correct the damaged


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strand, or (b) double strand breaks (DSB) occur in which two strands are nicked. The latter is particularly dangerous as DSBs may not be internally repaired and may result in genome rearrangements within a cell. As already noted, the much smaller frequencies for DSBs occurrence, compared to those for SSBs, makes double strand breaks very rare in cells. However, it is worth noting that DSBs are cytotoxic in nature and, therefore, even a few can induce cells death. About a decade ago experiments in which low-energy (few eV) electrons were made to collide with DNA (in dry form) helped establish a new paradigm: even slow electrons may induce strand breakages due to formation of transient molecular anions3 which dissociate on timescales of a few vibrational periods. Subsequent experiments were conducted in the biologically-relevant aqueous state;13 these demonstrated damage to DNA induced by slow electrons and .OH-radicals that were generated, in situ, in a laser-induced plasma. The plasma resulted from irradiation of water (in which plasmid DNA was suspended) by high intensity (112 TW cm-2), 30 fs long pulses of 800 nm light.13 These low-energy particles induced transformation of supercoiled DNA into relaxed DNA, a signature of SSBs, or even into linear DNA, a signature of DSBs. Judicious addition of varying concentrations of electron and .OH scavengers established that .OH-radicals have a fourfold higher propensity than slow electrons to induce strand breakages. Experiments conducted at longer wavelengths (1.35, 2.2 μm) served to delineate the role played by .OH-radicals in inducing strand breaks in DNA under physiological conditions.14 Results from these experiments strengthened the shift in paradigm that plasma effects in aqueous biological media are mediated via radiation chemistry triggered upon photolysis of water and not by direct interaction of DNA with the strong laser field, nor by multiphoton effects. The observation that nicks may be induced by plasma-induced photolysis of


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water is strongly indicative of biophotonics implications beyond studies of DNA per se, and opening new vistas for probing the effects of low energy radiation on living matter under physiological conditions. However, proper insights into how low-energy electrons and .OHradicals induce DNA damage continue to remain elusive. Electron-induced strand breakages were initially thought to result from dissociative electron attachment of few-eV electrons to nucleobases/nucleotides in water solutions15 but recent experiments16 have cast doubts on this mechanism. .OH-induced strand breakages, especially DSBs, are thought to result from collisional abstraction of a deoxyribose H-atom from the DNA backbone,17 but the problem of understanding .OH reactivity in aqueous media remains intractable due to the dynamics being dependent not only on the energy of each .OH-radical but also on the arrangement and conformations of all neighboring H2O molecules. In spite of the lack of proper insights and continuing theoretical challenges, it remains important to make use of new experimental ways to probe low-energy pathways to DNA damage. The importance of utilizing optical methods has been noted not only from the viewpoint of basic physics but also to shed new light into how cancers are caused and to help develop newer radiotherapy strategies for cancer treatment.18 This is primarily because detrimental dose distributions within proximate healthy tissue irradiated by -radiation - one of the major difficulties in contemporary radiotherapy - might be avoided by use of laser irradiation.19 Laser pulses, particularly in the infrared region, can readily be spatially confined to volumes that are some orders of magnitude smaller than what is possible to attain using contemporary clinical radiation sources: it, therefore, becomes possible to laser-irradiate cancerous tissue over very small spatial extents, thereby ensuring that proximate healthy tissue remains unaffected by laser light. In this connection, there is some evidence that near-infrared laser irradiation may yield


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essentially the same radiation dosage in the radiolysis of water as that obtained using very energetic γ-radiation.20 We report here results of a simple, user-friendly and inexpensive experiment based on a nanosecond Nd:YAG laser to probe plasma-induced DNA damage over an extremely small spatial volume (~few hundred μm3). The use of low-intensity nanosecond laser pulses affords a new perspective in that we can probe DNA damage that is induced by thermalized electrons and .OH-radicals.

It is known that the initial energy with which an electron is formed (in the

ionization process within the plasma) is thermalized in the course of hydration on timescales that are considerably shorter (~1 ps) than our pulse duration (6 ns).21 Thermalization of the .OHradical also occurs on much shorter timescales (~200 ps) than our laser pulse duration.22 In our experiments we have discovered that such thermal-energy electrons and .OH-radicals also induce strand breakages in DNA, including DSBs, as well as possible base oxidation/degradation. The latter is deduced by us by means of absorption spectroscopy while for the former we rely on agarose gel electrosphoresis. We have succeeded in determining the time-evolution of the damage over periods up to 330 seconds. Our results indicate that it takes barely 30 s for damage to occur. Furthermore, we have discovered that addition of physiologically relevant concentrations of a salt (NaCl) has an effect on strand breakages, including inhibiting them. We rationalize such inhibition using simple electrostatic considerations.

Methods We used 6 ns pulses (50 mJ) of 1064 nm Coherent light (Q-smart450, Quantel, France) to illuminate a highly collimated jet of nuclease-free water in which plasmid DNA (pUC19,


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Thermo Fisher Scientific, India; concentration 19 μg/ml) was suspended (Fig. 1). The incident laser intensity was adjusted so as to be near the threshold for optical breakdown, with focusing conditions (using a f = 100 mm lens) such that the Rayleigh range (1.4 mm with 1064 nm light) was larger than the width of the liquid jet (640 μm), ensuring that the aqueous plasmid DNA is exposed to the most uniform portion of the incident light. The extremely thin target places demands on laser pointing stability; Figure 1 also shows typical images of the laser-induced plasma; these were monitored to confirm uniformity in intensity and spatial dimensions of the laser focal volume. Emission spectra of the plasma were also monitored using 180º collection geometry. A f = 50 mm lens focused this collected signal to an optical fiber which was then fed into an echelle based spectrograph coupled with Intensified-Charge Coupled Device (ICCD, Andor, Ireland). The characteristic feature that makes our system particularly useful for spectroscopy measurements under our conditions is: wide spectral band-pass (230-850 nm) with high resolution (0.05 nm) in a single scan. Wavelength calibration of the system was performed using NIST certified lamps (Ocean Optics, USA).


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Figure 1. Schematic depiction of our experimental set-up in which focused light pulses (1064 nm wavelength), of 6 ns duration, from an Nd:YAG laser (10 Hz repetition rate) intersect a jet of water in which plasmid DNA (pUC19) is suspended. (a) The laser-DNA interaction geometry. The liquid jet is much smaller in size than the Rayleigh range of the laser beam, ensuring irradiation is at a constant intensity. The liquid flow-rate in our closed-cycle system is 1 ml s-1. (b) Images of laser-induced plasma over different laser shots, showing a high degree of laser pointing stability. (c) A typical 3-D spatial profile of the breakdown plasma. Spectrometers used to monitor emission and absorption spectra are also shown (see text).

Absorption measurements on laser-irradiated samples were carried out using a doublebeam, double-monochromator, ratio-recording UV/Vis/NIR spectrophotometer (Lambda 950, Perkin Elmer, USA) operating over the 175 - 3300 nm range with UV-Vis resolution of ≤ 0.05 nm and NIR resolution of ≤ 0.2 nm. Detection was by a photomultiplier tube over the UV/Vis range and an Indium Gallium Arsenide and Lead Sulfide detector for the NIR range. The lamp source used was deuterium for UV and tungsten-halogen for Vis/NIR.


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After laser irradiation of the plasmid DNA, we utilized standard gel electrophoresis to enable separation. Size determination was accomplished with reference to commercially available DNA ladders containing linear fragments of known length. For our electrophoresis measurements, 0.8% of agarose gel was prepared with ethidium bromide (0.5 mg/ml). 3 μl of 6X DNA loading dye (containing bromophenol blue and xylene cyanol FF) was added to 30 μl of plasmid sample before loading the gel. Electrophoresis was carried out using a 120 V electrical supply in 0.5X TBE (Tris-Borate-EDTA) buffer. Measurements were also made on plasmid DNA that was not exposed to the laser light; these served as control and enabled us to check sample purity in each set of experiments. Gel documentation system from Syngene was used to capture the gel images and ImageJ was used to process and analyze these images. Very occasionally, the control experiments revealed evidence for strand breaks in the sample prior to laser irradiation. These were induced as a result of inadequate sample preparation procedures (temperature, vigorous handling). Data from such (rare) occasions was not considered.

Results As mentioned above, we have used 6 ns long pulses of 1064 nm light to irradiate water in which plasmid DNA (pUC19) is suspended. The laser-induced breakdown of water results in excitation, ionization, and dissociation of H2O molecules, yielding species like H2O*, H2O+, .OH, .OH*,

and slow electrons. Collisions between electronically excited states of water and ionized

water molecules also leads to formation of .OH radicals23 which are long-lived (μs lifetimes).24 Within the plasma, pre-hydrated electrons may also attach to H2O, forming an H2O- anion that,


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after a few hundred attoseconds,25 dissociates into .OH and H-. The presence of peaks attributable to O, O+ and .OH in the emission spectrum measured from the region of optical breakdown (Fig. 2) provide confirmatory signatures of the occurrence of ionization and dissociation of water that we monitor. After irradiation for varying periods (30-330 s), the DNA samples were separated offline - by means of gel electrophoresis. Post-separation, the gel was stained with a DNA

Figure 2. Emission spectra of laser-induced plasma showing peaks due to (a) H and .O atoms, (b) .OH radicals, and (c) O+ ions. The inset identifies the respective transitions.

binding fluorescent dye (ethidium bromide) that allowed quantification of the amounts of DNA in different forms. Some typical results are shown in Fig. 3. Prior to laser irradiation, pUC19 is almost entirely (~92%) in the supercoiled (S) state, with only about 8% in the relaxed (R) state. After only 30 s of irradiation, the S component disappears almost totally (S~1%), to be replaced


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mostly by the R component (R~75%) with a significant fraction (~24%) assuming the linear (L) form. The L form indicates the occurrence of DSBs. Surprisingly, DSBs seem to be readily

Figure 3. Upper panel: A typical gel image supercoiled (S), relaxed (R), and linear (L) forms of plasmid DNA (pUC19) without laser irradiation (0 s) and upon exposure, over different time intervals, to 1064 nm light pulses of 6 ns duration and 50 mJ power. Lower panel: Corresponding data in the presence of 50 mM NaCl salt. Irradiation time refers to the time for which the liquid jet is exposed to laser light.

induced by thermalized electrons and/or .OH-radicals within a very short laser irradiation time of 30 s. By 60 s, the R and L components are seen to be almost equally prominent. The degradation of the plasmid DNA is obvious in the images of the R and L lines (Fig. 3) after about 240 s of laser irradiation. This degradation is most likely due to thermal effects. We also conducted experiments in which a salt, NaCl, was added in physiologically relevant concentrations (~50


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mM) to the water+DNA solution. This resulted in a significant and unexpected alteration of the overall damage dynamics, as depicted in Fig. 4. Now, the S form is seen to reduce monotonically with irradiation time, with its fraction decreasing down to S~12% at 270 s. The proportion of R form increases with time, becoming as large as R~80% at 270 s. The L form remains totally absent until 240 s; by 270 s it is ~8% and increases to ~20% by 330 s. Gel images (Fig. 3) indicate that the extent of thermal degradation is less pronounced than in the salt-less case. The temporal dynamics depicted in Figs. 3,4 show that, in the absence of salt, (i) SSBs are induced both prominently and fast, (ii) DSBs are also readily induced, and on fast timescales, and (iii) no supercoiled form survives after barely a minute’s irradiation. Moreover, addition of small (but physiologically relevant) concentration of NaCl is seen to significantly inhibit the propensity for plasma-induced single- and double-strand breakages. Indeed, in the presence of salt DSBs require irradiation for about 270 s before they manifest themselves. Concomitantly, the percentage of supercoiled DNA decreases monotonically, but it persists for significantly longer time periods than in the absence of salt; indeed, the S form is seen in the gel images (at the 10% level) for irradiation times as long as 330 s. It is pertinent to note here that addition of NaCl does not seem to produce any additional lines in the gel images that might be indicative of DNA fragmentation in these experiments.


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Figure 4. Percentages of different forms of DNA as a function of laser irradiation time without NaCl (upper panel) and with 50 mM NaCl (lower panel).

Discussion Before proceeding to rationalize our observations, we first consider whether damage to our plasmid DNA might possibly be due to conventional photoexcitation rather than plasmainduced effects? It is known that DNA’s maximum linear absorption of photons occurs at 260 nm wavelength, and that such photoexcitation is a likely cause of lesions.26 At 1064 nm wavelength, 4-photon excitation would be necessary to induce photo-damage. Although we


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expect that multiphoton effects are unlikely at the laser intensity values in these series of experiments we, nevertheless, made measurements at other levels of incident laser energy (up to 100 mJ) while keeping focusing and other conditions uniform. Gel images obtained at higher laser energy confirmed that the propensity for strand breakages in the absence of salt remains essentially the same, offering evidence against multiphoton effects playing a role in the DNA damage dynamics. Earlier work has also addressed the question as to whether strand breakages are induced by low-energy electrons or OH-radicals, both of which are constituents of the plasma that is created in our experiments.13,14 Measurements carried out in the presence of varying concentrations of electron- and OH-scavengers have confirmed that the latter are four times more likely to be responsible for SSBs and DSBs.14 In the present experiments, our conditions (mainly the long laser pulse duration) ensures that both these plasma constituents have sufficient time to become thermalized. In the case of electrons, extensive computer simulations have been carried out27 that have enabled yields to be deduced of various strand breaks and base damage via the dissociative electron attachment (DEA) process.3 As already noted, recent work16 has questioned the premise that DEA is responsible for strand breakages in DNA. Notwithstanding this, we note here that values of DEA cross sections for all four DNA bases are found to be close to zero for electron energies below 1 eV.27 Hence, not much DEA is to be expected with thermal energy electrons. This is consistent with our observations that, in the overwhelming majority of gel images that we accumulated in the course of our experiments, no signatures of SSB or DSB were seen in un-irradiated samples. We attribute the majority of strand breakage events in our experiments to be induced by thermal-energy .OH radicals. On the basis of earlier work conducted using longer wavelength laser light14 we estimate that SSBs and DSBs induced due to


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temperature effects account for only a minority (~10%) of strand breakages observed in our experiments.

Figure 5. An electrostatic model depicting salt cations forming a Na+ nanostructure that acts as a shield around the negatively charged DNA backbone, protecting it from .OH-radicals and electrons present in the plasma surrounding the DNA.

It is known that nucleic acids possess a high surface charge density that leads to the formation of an “ion atmosphere” around DNA which plays an important role in determining its physico-biological properties.28 From a structural viewpoint, DNA’s double-helix shape is formed by two linear sugar-phosphate backbones that run opposite each other and are twisted to form a double helical structure. The phosphate groups in the backbone are negatively charged whereas the four nucleic bases are positively charged. Though the charges are balanced and, on the whole, DNA is a neutral hydrophilic molecule, the negative charges of the phosphates on the outside provide a protective “envelope” for the bases. But these negative charges can also bind with external metal ions so as to form a nanoscale electrostatic shield that adds to the protection


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of the base pairs. In the absence of external cations, the phosphate groups of the backbone are almost completely ionized; repulsion between the negative charges induces strain within the helix, making it susceptible to strand breaks upon interactions with plasma constituents like low energy electrons and .OH-radicals in our experiments. However, when external cations, like Na+ from salt, are introduced, they are able to bind to the negatively charged phosphate, forming the protective electrostatic shield around DNA that we refer to. It is this Na+ nanostructure that potentially helps to inhibit strand breakages. Figure 5 depicts a schematic representation of this electrostatic environment, in which a Na+ nanostructure acts as a shield that protects the DNA. A consequence of these simple electrostatic considerations lends itself to a simple experimental test. Inhibition of DNA damage upon addition of cations like Na+ would be expected to manifest itself as an increase in DNA melting temperature. When DNA is heated, the double-stranded DNA (dsDNA) unwinds and separates into single-stranded (ssDNA) by breaking the hydrogen bonds between the bases (A=T and G≡C). Biologists refer to this as DNA denaturation and it can be monitored by measuring absorbance in the region of 260 nm.29 In general, absorbance around 260 nm is expected to increase as the DNA becomes denatured. However, this is not what is measured in our experiments. Figure 6 shows absorbance in the proximity of 260 nm with and without salt but without laser irradiation. We have measured the ratio of the absorbance peak to the background (taken to be at 318 nm) to be 11.2 without laser light. Most significantly, upon laser irradiation, the 260 nm peak decreases in our experiments. The ratio, in the absence of NaCl reduces from 11.2 to ~3 within 30 s and eventually becomes about 1.5 after 240 s. When NaCl is added, the reduction in the ratio is less marked: after 30 s irradiation the ratio falls to only about 8.5 from 11.2 for pristine DNA (Fig. 6). Thereafter, the


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ratio to ~4.5 after 300 s, a factor of three larger than in the case without NaCl. This factor is likely to be a measure of

Figure 6. Time evolution of the absorbance ratio for laser-irradiated pUC19 plasmid DNA without NaCl (red circles) and with NaCl (black triangles). The ratio is absorption at the peak to that at 318 nm (taken to be the background). The inset shows absorbance spectra of pUC19 prior to laser exposure; addition of 50 mM NaCl only marginally affects the 260 nm absorbance. Upon laser irradiation, the overall absorbance ratio of pUC19 with salt decreases very substantially. Irradiation time of zero implies no laser irradiation.

electrostatic protection against damage introduced into the DNA’s ion atmosphere by Na+ ions. This is in conformity with the predictions of our electrostatic model wherein the plasma constituents - electrons and .OH-radicals – act as damage-inducing agents. But why does the ratio of the 260 nm peak to background decrease? As already noted, it cannot be ascribed to strand breakages as these would be expected to manifest themselves in an increase of the ratio. As is well known, single stranded DNA has higher absorption at 260 nm compared to double-


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stranded and, hence, it would be expected that the ratio would increase with irradiation time. But, it has been established that chemical modification of DNA induced by UV-B irradiation30 results from oxidation of the bases, specifically guanine, and this leads to a decrease in the intensity of the 260 nm peak. UV-A radiation also induces photo-oxidation of, mainly, guanine residues of cellular DNA. UV-C does not reach the earth’s surface and does not exhibit harmful effects.31 In order to further probe into the physics that underlies the reduction in the spectral intensity at 260 nm, it would be of interest to carry out supplementary experiments involving immunoslot blot or enzymatic detection;32 such experiments may provide further confirmation of the presence of plasma-induced oxidative DNA damage. Our observations may open opportunities of further exploitation in terms of using different salts to electrostatically engineer the propensity for protection against low-energy radiation.33 Our results seem to offer indications that plasma-induced effects in biological systems may have wider utility than initially thought not only in studying the effects of very lowenergy radiation damage but also to explore processes that inhibit radical-induced damage in living matter.

Conclusions Low-power nanosecond-long pulses of 1064 nm laser light have been used to create plasma in an aqueous solution of plasmid DNA (pUC19). Our use of a liquid jet that intersects the laser beam neatly circumvents problems created by plasma effects at surfaces and enables spectroscopic monitoring of the laser-induced plasma. The long duration of the laser pule ensures thermalization of plasma constituents like electrons and .OH-radicals. These thermal energy


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particles are found to induce strand breakages in DNA, including double strand breaks, and possible base oxidation/degradation. Time-evolution measurements reveal that it takes barely 30 s for damage to DNA to occur. Addition of physiologically relevant concentrations of a salt (NaCl) significantly inhibits such damage. We rationalize such inhibition using a simple electrostatic model. Our results on plasma-induced DNA damage suggest implications beyond studies of DNA; our findings may open new vistas for developing new experimental insights into how low-energy radiation affects living matter under physiological conditions.

AUTHOR INFORMATION

Corresponding Author *[email protected]

ORCID Abdul Ajees Abdul Salam: 0000-0002-3377-3048 Parinda Vasa: 0000-0002-3182-0736 Santhosh Chidangil: 0000-0002-2973-6834 Deepak Mathur: 0000-0002-2322-6331

Authors’ Contributions VKU, AAAS, SC and DM designed the research. UN, VKU, AAAS performed the experiments. PV was responsible for the electrostatic model. All authors wrote and reviewed the manuscript.


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NOTES The authors declare no competing financial interest.

ACKNOWLEDGMENTS We gratefully acknowledge Dr. G. Arunkumar and Dr. J. Anitha of the Department of Virus Research, MAHE for facilitating the gel electrophoresis measurements. DM thanks the Science and Engineering Research Board for financial support through the award of the J. C. Bose National Fellowship (SR/S2/JCB-29/2006). We gratefully acknowledge financial support from the joint MAHE and FIST program of the Government of India (SR/FST/PSI-174/2012). We also thank the Board of Research in Nuclear Sciences (BRNS) for financial support (34/14/04/2014-BRNS). AAAS acknowledges the research grant provided by MAHE (MAHE/REG/TD/DAMP-(T)) under the fellowship program offered by Manipal FAIMER Institute (M‐FIILIPE). PV acknowledges support from the Department of Biotechnology award to

the

Wadhwani

Research

Centre

for

Bioengineering

at

IIT

Bombay

(BT/INF/22/SP23026/2017).

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Strong Strand Breaks in DNA Induced by Thermal Energy Particles

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