Determination of DNA structural detail using radioprobing

Abstract

Purpose: To put radioprobing into context as a relatively new method of determining structural detail in deoxyribonucleic acid (DNA), and to review its use since first proposed in 1997. The key feature of the method is that, by experiment or simulation, a radionuclide such as iodine-125 (¹²⁵I) is placed near the DNA at a known point relative to the DNA base sequence, and the number of resulting strand breaks in each nucleotide is determined. As the intensity of damage declines consistently with distance from the radionuclide, relative distances between the emitter and the nucleotides can be deduced, and hence potentially the topology or structural detail of the DNA. For simulation, appropriate software includes a Molecular Dynamics package, analysis and visualization tools, and a Monte Carlo track structure program. Conclusions: A review of published work and our own recent unpublished studies have shown that radioprobing is sufficiently sensitive and consistent to determine structural detail such as internal folding topology and flexing behavior, and can be applied to DNA or a DNA-protein complex in an approximation to its normal biological environment.     

The Auger effect in physical and biological research

Purpose: The paper reports on progress in physics of radiationless transitions and new Auger spectra of ¹²⁵I and ¹²⁴I. We report progress in Monte Carlo track structure simulation of low energy electrons comprising majority electrons released in decay most Auger emitters.

Materials and methods: The input data for electron capture (EC) and internal conversion(IC) were obtained from various physics data libraries. Monte Carlo technique was used for the simulation of Auger electron spectra. Similarly, electron tracks were generated using Monte Carlo track structure methods.

Results: Data are presented for the EC, IC and binding energy (BE) of radionuclides ¹²⁴I and ¹²⁵I. For each of the radionuclides ¹²⁵I and ¹²⁴I some examples of electron spectra of individual decays are given. Because most Auger electrons are low energy and short range, data and a short discussion are presented on recent Monte Carlo track structure development in condensed media and their accuracy.

Conclusions: Accuracy of electron spectra calculated in the decay of electron shower by Auger emitting radionuclides depends on availability of accurate physics data. There are many gaps in these libraries and there is a need for detailed comparison between analytical method and Monte Carlo calculations to refine the method of calculations. On simulation of electron tracks, although improved models for sub-keV electron interaction cross sections for liquid water are now available, more experimental data are needed for benchmarking. In addition, it is desirable to make data and programs for calculations of Auger spectra available online for use by students and researchers.     

Determination of DNA structural detail using radioprobing

PURPOSE: To put radioprobing into context as a relatively new method of determining structural detail in deoxyribonucleic acid (DNA), and to review its use since first proposed in 1997. The key feature of the method is that, by experiment or simulation, a radionuclide such as iodine-125 ((125)I) is placed near the DNA at a known point relative to the DNA base sequence, and the number of resulting strand breaks in each nucleotide is determined. As the intensity of damage declines consistently with distance from the radionuclide, relative distances between the emitter and the nucleotides can be deduced, and hence potentially the topology or structural detail of the DNA. For simulation, appropriate software includes a Molecular Dynamics package, analysis and visualization tools, and a Monte Carlo track structure program. CONCLUSIONS: A review of published work and our own recent unpublished studies have shown that radioprobing is sufficiently sensitive and consistent to determine structural detail such as internal folding topology and flexing behavior, and can be applied to DNA or a DNA-protein complex in an approximation to its normal biological environment.     

Monte Carlo-simulated Auger electron spectra for nuclides of radiobiological and medical interest – a validation with noble gas ionization data

Abstract

Purpose: To further validate Monte Carlo calculation codes simulating cascades of Auger electron transitions in radionuclides that decay by electron capture or internal conversion. In particular, the need for an appropriate kinetic energy determination of the Auger electrons emitted from multiple-ionized atoms as well as the consideration of shake-off electrons would be investigated implicitly.

Methods: Charge distributions of noble gases after photoionization for different photon energies were calculated and compared with experimental data from the literature. In addition, new electron emission spectra were generated for ^99mTc and ¹²³I.

Results: By including strict energy book-keeping and allowing shake-off electrons, the agreement between experimentally detected charge distributions and Monte Carlo simulations was very good. On this basis, the number of emitted electrons per decay was found to be between 1 and 17 with a mean of 4.0 for ^99mTc and between 1 and 26 with a mean of 7.4 for ¹²³I.

Conclusions: Because of the good agreement with the experimental findings, the validation can be considered to be successful.     

Calculated strand breaks from 125I in coiled DNA

Purpose: DNA single strand breaks (SSB) and double-strand breaks (DSB) induced by Auger electrons from incorporated ¹²⁵I decay were calculated using a B-DNA model to assess contributions from direct and OH damage and effects of higher-order structure. Three decay sites, linker DNA, nucleosome, and two adjacent nucleosomes, were assessed and compared to experimental data.

Method: A Monte Carlo track structure code for electron was used to track electrons, OH and H radicals through linear and a higher-order model of B-DNA. Direct and indirect DNA hits were scored and used to determine SSB and DSB.

Results: The three different ¹²⁵I decay locations produced different number of DSBs and fraction of radical damage. The average number of DSB per ¹²⁵I decay was 0.83, 0.86 and 1.33, respectively, for the three sites. OH radical attack contributed to or exclusively caused 70%, 57%, and 50%, of the DSBs located in the entire model. When only 10 base pairs on either side of the incorporation site were considered, radical damage contributions were 40%, 25% and 67%, respectively. Locations distant from the site of incorporation, however, consistently yielded 70–80% of the DSB from radical attack.

Conclusions: Coiling of DNA can greatly change both the absolute number of DSB per incorporated ¹²⁵I decay and the relative contributions of radical damage to the local site of decay and, to a lesser extent, the average over all DNA. Higher order structure only slightly affects the number and quality of DNA damage to distant locations, which is mostly from radical attack.     

Track structure,radiation quality and initial radiobiological events: Considerations based on the PARTRAC code experience

Abstract

Purpose: The role of track structures for understanding the biological effects of radiation has been the subject of research activities for decades. The physics that describes such processes is the core Monte Carlo codes, such as the biophysical PARTRAC (PARticle TRACks) code described in this review, which follow the mechanisms of radiation-matter interaction from the early stage. In this paper a review of the track structure theory (and of its possible extension concerning non-DNA targets) is presented.

Materials and methods: The role of radiation quality and track structure is analyzed starting from the heavy ions results obtained with the biophysical Monte Carlo code PARTRAC (PARticles TRACks). PARTRAC calculates DNA damage in human cells based on the superposition of simulated track structures in liquid water to an ‘atom-by-atom’ model of human DNA. Results: Calculations for DNA fragmentation compared with experimental data for different radiation qualities are illustrated. As an example, the strong dependence of the complexity of DNA damage on radiation track structure, and the very large production of very small DNA fragments (lower than 1 kbp (kilo base pairs) usually not detected experimentally) after high LET (high-Linear Energy Transfer) irradiation is shown. Furthermore the possible importance of non-nuclear/non-DNA targets is discussed in the particular case of cellular membrane and mitochondria.

Conclusions: The importance of the track structure is underlined, in particular the dependence of a given late cellular effect on the spatial distribution of DNA double-strand breaks (DSB) along the radiation track. These results show that the relative biological effectiveness (RBE) for DSB production can be significantly larger than 1. Moreover the cluster properties of high LET radiation may determine specific initial targets and damage evolution.     

Heterogeneity in Vascular Architecture and Heat-induced Vascular Damage of Human Melanoma Xenograft Lines Established from Different Lesions in the Same Patient

Abstract

Purpose: With the advent of magnetic resonance imaging (MRI)-guided radiation therapy it is becoming increasingly important to consider the potential influence of a magnetic field on ionising radiation. This paper aims to study the effect of a magnetic field on the track structure of radiation to determine if the biological effectiveness may be altered.

Methods: Using the Geant4-DNA (GEometry ANd Tracking 4) Monte Carlo simulation toolkit, nanodosimetric track structure parameters were calculated for electrons, protons and alpha particles moving in transverse magnetic fields up to 10 Tesla. Applying the model proposed by Garty et al., the track structure parameters were used to derive the probability of producing a double-strand break (DSB).

Results: For simulated primary particles of electrons (200 eV–10 keV), protons (300 keV–30 MeV) and alpha particles (1–9 MeV) the application of a magnetic field was shown to have no significant effect (within statistical uncertainty limits) on the parameters characterizing radiation track structure or the probability of producing a DSB.

Conclusions: The null result found here implies that if the presence of a magnetic field were to induce a change in the biological effectiveness of radiation, the effect would likely not be due to a change in the track structure of the radiation.     

Simulation of ionisation clusters formed in nanometric volumes of the deoxyribose-substitute tetrahydrofuran

Abstract

Purpose: To investigate the implications of using interaction cross sections of liquid water for the target volume when studying radiation action at the DNA level by particle track structure simulations.

Materials and methods: Absolute interaction cross sections for low energy electrons between 20 eV and 1 keV were measured for tetrahydrofuran (THF), which is a substitute for deoxyribose. From these data a complete interaction cross section data set was derived and integrated in our PTB Track structure Monte Carlo code ‘PTra’. Simulations of electron track structure in THF and water were performed and ionisation cluster size distributions in nanometric target volumes were determined. From these a nanodosimetric estimate for the probability to produce a double strand break was derived.

Results: The probability distribution of ionisation cluster sizes was found to be shifted towards smaller values for a THF-filled target as compared to a water-filled one. For all electron energies investigated, the nanodosimetric estimates for double-strand break probability in the THF-filled target have lower values than for a target of liquid water.

Conclusion: The preliminary results indicate that simulations based on cross sections of water would overestimate the initial direct radiation damage to the DNA.     

Stochastic modelling of DSB repair after photon and ion irradiation

Abstract

Purpose: To test the stochastic model for DNA double-strand break (DSB) repair via non-homologous end joining (NHEJ) implemented in the Monte Carlo code PARTRAC (PARticle TRACks) against measured repair kinetics after nitrogen ion and ⁶⁰Co γ reference irradiation.

Material and methods: By combining Monte Carlo track structure calculations with multi-scale models of cellular DNA, yields of DSB are calculated for N ion and ⁶⁰Co γ-irradiation. The NHEJ model in PARTRAC is used to determine rejoining kinetics of the DNA ends and DNA fragment distributions after certain repair times. Model parameters are adapted to the measured rejoining kinetics for the different radiation types.

Results: DSB rejoining kinetics after low- and high-linear energy transfer (LET) irradiation have been reproduced after refinements of the DNA repair model, in particular by considering an ongoing production of detectable DSB in the initial phase, e.g., by enzymatic processing of labile sites, and by assuming a limited availability of repair enzymes needed for processing complex lesions during the slow repair phase.

Conclusions: The need for certain model refinements suggests mechanisms that may significantly contribute to the DSB rejoining kinetics during both initial and later phases of NHEJ.     

基于实验与蒙特卡罗模拟法探究125I粒子间剂量衰减

目的 研究多颗¹²⁵I放射性粒子间剂量衰减规律.方法 利用Geant 4软件包进行蒙特卡罗模拟单颗粒子和多颗粒子周围剂量分布,将模拟结果与TG43-U1报告中推荐剂量计算方法所得结果进行对比,并利用实验测得数据验证蒙特卡罗模拟结果.结果 单颗粒子周围剂量分布的蒙特卡罗模拟结果与TG43-U1计算和实验结果差值分别在±3%和±5%以内.多颗粒子的蒙特卡罗模拟结果对比TG43-U1线性叠加结果,粒子间剂量衰减为3.8%~13.2%,平均剂量衰减为7.2%,实验所得结果与蒙特卡罗模拟结果差值在6%以内.结论 空间中存在多颗放射性¹²⁵I粒子时,由于粒子间的相互影响导致7%左右的剂量衰减,其最大值可超过13%,在人体组织中剂量衰减值会更大.因此,利用TG43-U1方法进行线性叠加计算临床中的剂量分布不够精确.     

Abstracts

Abstract

Purpose: To model interaction cross sections and energy loss for carbon projectiles C⁰–C⁶⁺ of 1–10⁴ keV/u (u: atomic mass unit) in water.

Materials and methods: The classical trajectory Monte Carlo method was used to calculate the ionisation and charge-transfer cross sections. The excitation cross sections were scaled from proton data using equilibrium charges determined from the charge-transfer cross sections. Energy loss was obtained from the singly differential cross sections, and ionisation potentials of the target and projectile.

Results: The calculated total ionisation cross sections are consistent with measured data, while the calculated electron-capture cross sections are larger than experimental data by a factor of 3. By scaling the latter to the measured data, the cross sections were made consistent with these data for 1–10 keV/u energies. The present stopping cross sections agree well with experimental data below 10 keV/u, and with other model calculations above 2 MeV/u. Deviation from the latter is found where electron capture is competitive with ionisation, and also arises from different energy-transfer calculations.

Conclusions: In this paper we report our efforts in the developments of full slowing-down Monte Carlo track structure calculations for carbon ions. Further development and refinement of the model are currently underway.     

Geant4 hadronic physics for space radiation environment

Abstract

Purpose: To test and to develop Geant4 (Geometry And Tracking version 4) Monte Carlo hadronic models with focus on applications in a space radiation environment.

Materials and methods: The Monte Carlo simulations have been performed using the Geant4 toolkit. Binary (BIC), its extension for incident light ions (BIC-ion) and Bertini (BERT) cascades were used as main Monte Carlo generators. For comparisons purposes, some other models were tested too. The hadronic testing suite has been used as a primary tool for model development and validation against experimental data.

Results: The Geant4 pre-compound (PRECO) and de-excitation (DEE) models were revised and improved. Proton, neutron, pion, and ion nuclear interactions were simulated with the recent version of Geant4 9.4 and were compared with experimental data from thin and thick target experiments.

Conclusions: The Geant4 toolkit offers a large set of models allowing effective simulation of interactions of particles with matter. We have tested different Monte Carlo generators with our hadronic testing suite and accordingly we can propose an optimal configuration of Geant4 models for the simulation of the space radiation environment.     

A model of the cell nucleus for DNA damage calculations

Abstract

Aims: Development of a computer model of genomic deoxyribonucleic acid (DNA) in the human cell nucleus for DNA damage and repair calculations. The model comprises the human genomic DNA, chromosomal domains, and loops attached to factories.

Material and methods: A model of canonical B-DNA was used to build the nucleosomes and the 30-nanometer solenoidal chromatin. In turn the chromatin was used to form the loops of factories in chromosome domains. The entire human genome was placed in a spherical nucleus of 10 micrometers diameter. To test the new target model, tracks of protons and alpha-particles were generated using Monte Carlo track structure codes PITS99 (Positive Ion Track Structure) and KURBUC. Damage sites induced in the genome were located and classified according to type and complexity.

Results: The three-dimensional structure of the genome starting with a canonical B-DNA model, nucleosomes, and chromatin loops in chromosomal domains are presented. The model was used to obtain frequencies of DNA damage induced by protons and alpha-particles by direct energy deposition, including single- and double-strand breaks, base damage, and clustered lesions.

Conclusions: This three-dimensional model of the genome is the first such model using the full human genome for the next generation of more comprehensive modelling of DNA damage and repair. The model combines simple geometrical structures at the level of domains and factories with potentially full detail at the level of atoms in particular genes, allowing damage patterns in the latter to be simulated.     

Charge build-up during decay of DNA-incorporated 123/125I: Consequences for labeled molecular structures

Abstract

Purpose: To further elucidate the mechanisms behind the strong biological effectiveness of DNA-incorporated Auger electron emitters ¹²³I and ¹²⁵I, which are mostly attributed to the shower of low-energy electrons released during the decay. A second, frequently mentioned cause can be seen in the charges accumulated during the Auger cascade on the decaying nuclide and its subsequent intra-molecular redistribution leading to a Coulomb explosion.

Methods: To assess the size of the charge and the dimensions of DNA damage thus determined, the first Auger cascade was simulated by Monte Carlo methods. The consequences of intra-molecular charge transfer in terms of structural molecular alterations were estimated by density functional theory (DFT) calculations and folding with the results of the Monte Carlo studies.

Results: Charge distributions of ¹²³I and ¹²⁵I were found to be very similar with values between +?1 and +?15 and a mean value of +?6.4. The molecules could tolerate charges up to +?5 (base), +?2 (nucleoside) and +?7 (nucleotide) without being destroyed.

Conclusions: The strong molecular DNA damage after ¹²³I and ¹²⁵I decay depends very much on the size of the DNA molecule involved in the calculation. In general, not every decay can be expected to lead to a Coulomb explosion.     

DNA double-strand breaks induced by decay of 123I-labeled Hoechst 33342: Role of DNA topology

Purpose: To determine double-strand-break (DSB) yields produced by decay of minor-groove-bound ¹²³I-labeled Hoechst 33342 (¹²³IEH) in supercoiled (SC) and linear (L) forms of pUC19 DNA, to compare strand-break efficiency of ¹²³IEH with that of ¹²⁵IEH, and to examine the role of DNA topology in DSB induction by these Auger electron emitters.

Materials and methods: Tritium-labeled SC and L pUC19 DNA were incubated with ¹²³IEH (0–10.9 MBq) at 4°C. After ¹²³I had completely decayed (10 days), samples were analyzed on agarose gel, and single-strand-break (SSB) and DSB yields were measured.

Results: Each ¹²³I decay in SC DNA produces a DSB yield of 0.18 ± 0.01. On the basis of DSB yields for ¹²⁵IEH (0.52 ± 0.02 for SC and 1.62 ± 0.07 for L, reported previously) and dosimetric expectations, a DSB yield of ～0.5 (3 × 0.18) per ¹²³I decay is expected for L DNA. However, no DSB are observed for the L form, even after ～2 × 10¹¹ decays of ¹²³I per μg DNA, whereas a similar number of ¹²⁵I decays produces DSB in ～40% of L DNA.

Conclusion: ¹²³IEH-induced DSB yield for SC but not L DNA is consistent with the dosimetric expectations for Auger electron emitters. These studies highlight the role of DNA topology in DSB production by Auger emitters and underscore the failure of current theoretical dosimetric methods per se to predict the magnitude of DSB.     

Effects of Anaesthetic Agent on [31P]NMR High-energy Phosphates and Regional Distributions of Intravascular Oxyhaemoglobin Saturations

Abstract

Purpose: The biological response of tissue exposed to radiations emitted by internal radioactivity is often correlated with the mean absorbed dose to a tissue element. However, experimental studies show that even when the mean absorbed dose to the tissue element is constant, the response of the cell population within the tissue element can vary significantly depending on the distribution of radioactivity at the cellular and multicellular levels. The present work develops theoretical models to simulate these observations.

Materials and methods: Two theoretical models were created to simulate experimental three-dimensional cell culture models with homogeneous and inhomogeneous tissue environments. The cells were assigned activities according to lognormal distributions of an alpha particle emitter or a monoenergetic electron emitter. Absorbed doses to the cell nuclei were assessed with point-kernel geometric-factor and Electron Gamma Shower version nrc (EGSnrc) Monte Carlo radiation transport simulations, respectively. The self- and cross-dose to individual cell nuclei were calculated and a Monte Carlo method was used to determine their fate. Survival curves were produced after tallying the live and dead cells.

Results: Both percent cells labeled and breadth of lognormal distribution affected the dose distribution at the cellular level, which in turn, influenced the shape of the cell survival curves.

Conclusions: Multicellular Monte Carlo dosimetry-models offer improved capacity to predict response to radiopharmaceuticals compared to approaches based on mean absorbed dose to the tissue.     

Antisense radiotherapy: targeting full‐size mdr1 mRNA with 125I‐labelled oligonucleotides

Purpose: Antisense radiotherapy is an approach based on the targeting of mRNA of specific genes by complementary oligonucleotide probes labelled with an Auger‐electron‐emitting radioisotope. Decay of the Auger emitter should specifically destroy the targeted mRNA while producing minimal damage to the rest of mRNA pool and the nuclear DNA. The feasibility of this approach was investigated by using full‐length human multidrug‐resistance gene (mdr1) mRNA as a target.

Materials and methods: Antisense oligonucleotides were labelled with [¹²⁵I] I‐dCTP by primer extension and annealed to target mRNA. Breaks in the target mRNA were analysed by denaturing polyacrylamide gel electriphoresis.

Results: The efficiency of ¹²⁵I‐labelled antisense oligonucleotides in producing RNA strand breaks was tested on short synthetic RNA and DNA targets. The position and specificity of ¹²⁵I‐induced breaks in the full‐length mRNA were then tested and compared with the cleavage of the target by RNase H. The distribution of the breaks in the longer mRNA is different from that in the short RNA targets, most likely due to a complex folding of RNA strands in the full‐length mRNA.

Conclusions: The authors posit that ¹²⁵I‐labelled antisense probes could be useful not only for targeting mRNA, but also as probes for mRNA folding in vivo.     

Microdosimetry of low-energy electrons

Abstract

Purpose: To investigate differences in energy depositions and microdosimetric parameters of low-energy electrons in liquid and gaseous water using Monte Carlo track structure simulations.

Materials and methods: KURBUC-liq (Kyushu University and Radiobiology Unit Code for liquid water) was used for simulating electron tracks in liquid water. The inelastic scattering cross sections of liquid water were obtained from the dielectric response model of Emfietzoglou et al. (Radiation Research 2005;164:202–211). Frequencies of energy deposited in nanometre-size cylindrical targets per unit absorbed dose and associated lineal energies were calculated for 100–5000 eV monoenergetic electrons and the electron spectrum of carbon K edge X-rays. The results for liquid water were compared with those for water vapour.

Results: Regardless of electron energy, there is a limit how much energy electron tracks can deposit in a target. Phase effects on the frequencies of energy depositions are largely visible for the targets with diameters and heights smaller than 30 nm. For the target of 2.3 nm by 2.3 nm (similar to dimension of DNA segments), the calculated frequency- and dose-mean lineal energies for liquid water are up to 40% smaller than those for water vapour. The corresponding difference is less than 12% for the targets with diameters ≥?30 nm.

Conclusions: Condensed-phase effects are non-negligible for microdosimetry of low-energy electrons for targets with sizes smaller than a few tens of nanometres, similar to dimensions of DNA molecular structures and nucleosomes.