An Approach to Modelling Radiation Damage by Fast Ionizing Particles

This paper presents a statistical approach to modelling thedamaging effects of radiation by fast heavy ionizing particlesin small biological structures such as enzymes, viruses, andsome cells. Irreparable damage is assumed to be caused by theoccurrence of ionizations within sensitive regions of a structure.For structures containing double-stranded DNA, one or more ionizationsoccurring within each strand of the DNA will cause inactivation;for simpler structures without double-stranded DNA a singleionization within the structure will be sufficient for inactivation.Damaging ionizations occur along tracks of primary irradiatingparticles or along tracks of secondary particles released atprimary ionizations. An inactivation probability is derivedfor each damage mechanism, and is expressed in integral formin terms of the radius of the biological structure (assumedspherical), the rate of ionization along primary tracks, andthe maximum energy for secondary particles. The performanceof each model is assessed by comparing results from the modelwith results derived from data from various experimental studiesextracted from the literature. For the simpler structures, wherea single ionization is sufficient for inactivation, the modelgives qualitatively promising results. However, for larger morecomplex structures containing double-stranded DNA, the modelrequires some further refinements.     

Comparison of nanodosimetric parameters of track structure calculated by the Monte Carlo codes Geant4-DNA and PTra

The concept of nanodosimetry is based on the assumption that initial damage to cells is related to the number of ionizations (the ionization cluster size) directly produced by single particles within, or in the close vicinity of, short segments of DNA. The ionization cluster-size distribution and other nanodosimetric quantities, however, are not directly measurable in biological targets and our current knowledge is mostly based on numerical simulations of particle tracks in water, calculating track structure parameters for nanometric target volumes. The assessment of nanodosimetric quantities derived from particle-track calculations using different Monte Carlo codes plays, therefore, an important role for a more accurate evaluation of the initial damage to cells and, as a consequence, of the biological effectiveness of ionizing radiation. The aim of this work is to assess the differences in the calculated nanodosimetric quantities obtained with Geant4-DNA as compared to those of the ad hoc particle-track Monte Carlo code 'PTra' developed at Physikalisch-Technische Bundesanstalt (PTB), Germany. The comparison of the two codes was made for incident electrons of energy in the range between 50 eV and 10 keV, for protons of energy between 300 keV and 10 MeV, and for alpha particles of energy between 1 and 10 MeV as these were the energy ranges available in both codes at the time this investigation was carried out. Good agreement was found for nanodosimetric characteristics of track structure calculated in the high-energy range of each particle type. For lower energies, significant differences were observed, most notably in the estimates of the biological effectiveness. The largest relative differences obtained were over 50%; however, generally the order of magnitude was between 10% and 20%.     

Particle track structure and its correlation with radiobiological endpoint

One of the possible ways to classify track structures is application of the conventional partition techniques of analysis of multidimensional data to the track structure. Using these cluster algorithms this paper attempts to find characteristics of radiation reflecting the spatial distribution of ionizations in the primary particle track. Absolute frequency distributions of clusters giving the mean number of clusters produced by radiation per unit of deposited energy have been computed for radiation of different qualities. The results were compared with the published experimental data of cell inactivation. For particular biological objects the critical properties of radiation correlating with the cell inactivation can be found and it seems that the occurrence of a cluster of at least four ionizations formed in a domain of approximately 2-3 nm correlates with the induction of double strand break.     

Cross-sections for the inactivation of ribonuclease by slow heavy ions.

Average cross-sections for inactivation of dry ribonuclease by H, He and N projectiles with energies between 100 eV and 10 keV are presented. The trend of the damage cross-sections with particle energy is consistent with the trend of the molecular quasi-elastic scattering and ionization cross-sections. A tentative radiation damage model is proposed in which single hit damage attributed to direct physical interactions by the primary and secondary radiations is supplemented by chemical action of the elastically scattered knock-on atoms which inactivate additional targets with an efficiency of 50-100%. Further experiments at energies below 100 eV are required for positive confirmation of the chemical efficiency. Free electrons (sub-ionizing) are chemically relatively innocuous (less than 10% efficiency). Saturation effects are satisfactorily accounted for in this model. Inactivation penetration depths measured in the microcrystalline enzyme agree with the theoretical predictions if a residual range of about 8 mug cm-2 is included. The mean energy expenditure to produce inactivation of a ribonuclease molecule is found to vary from 30 eV for 100 eV protons to 220 eV He and N ions.     

Intracellular visualization of precursor capsids in phage P22 mutant infected cells

The morphogenesis of the double-stranded DNA Salmonella phage P22 has been studied by electron microscopy of sections of wild type and mutant-infected cells. Previous work had shown that the precursor capsid structure that encapsidates DNA is a complex of two major protein species, the gene 5 coat protein and the gene 8 scaffolding protein. Scaffolding protein exits from the precursor capsid in coupling with DNA encapsidation and then recycles.No organized structures were seen in cells infected with amber mutants of the coat protein gene. Cells infected with an amber mutant of the scaffolding gene contained small numbers of aberrant particles, including empty petit capsids and giant nested or spiral shell structures.In cells infected with mutants blocked in DNA encapsidation (genes 1, 2, and 3) precursor capsids (proheads) accumulate amid the vegetative DNA and not along the membrane, as in phage T4. The proheads appear as double-shell structures about the size of mature phage, with the inner cell diameter about two-thirds the dimension of the outer shell.Mature phage and defective particles containing DNA form paracrystalline arrays within infected cells. Empty capsids, lacking both DNA and the inner shell of proheads, appear within the paracrystalline arrays of filled heads in cells infected with mutants blocked in head completion (genes 10 and 26). These empty capsids are presumably derived from filled but incomplete heads that have lost their DNA intracellularly.Use of temperature-sensitive mutants blocked in the encapsidation steps allowed visualization of the first filled heads upon shift to permissive temperature. These particles tended to appear at the edge of the DNA pool. Partially filled particles with dense central cores often were seen associated with the growing paracrystalline arrays, and they probably represented intermediates in encapsidation.These experiments, in conjunction with others, suggest that the scaffolding protein, which functions in prohead assembly and perhaps in DNA encapsidation, is organized into an inner shell within the precursor capsid.     

Intracellular forms of the parental human cytomegalovirus genome at early stages of the infective process.

Intracellular forms of human cytomegalovirus (HCMV) DNA molecules isolated from infected cells were examined by electron microscopy before and after the onset of viral DNA synthesis. In cells harvested before showing replicative forms of viral DNA, circular and concatemeric molecules were observed in addition to linear double-stranded molecules. The observation of circular, unbranched, unit-size molecules suggests that HCMV DNA has a repetitive sequence that is located at or near the termini and is exposed by exonuclease digestion within infected cells, and that single-stranded regions can complement each other to form circles. Linear molecules (unit size or smaller) and concatemers with replicative “eye” loops or forks were observed after DNA replication could be assumed to have begun. Viral DNA molecules with terminal loops were also observed. These structures indicate that an inverted repetition of the sequence may be present within the terminal region. The functions of these molecules and of circular molecules are unknown.     

Ultraviolet photobiology of Kilham rat virus and the absolute ultraviolet photosensitivities of other animal viruses: Influence of DNA strandedness,molecular weight,and host-cell repair

Kilham rat virus (KRV), which contains single-stranded DNA, is inactivated by ultraviolet (UV) radiation at 254 nm with a mean lethal dose that produces only 0.34 thymine-containing dimers per virion. These data, along with published values for the UV inactivation of numerous other mammalian viruses and of coliphages, with allowance being made for the molecular weights of the genomes, are consistent with a scheme of “weighted sensitivities.” The weighted sensitivities fall into three distinct classes. Class I, the most sensitive, includes viruses that contain single-stranded DNA and do not exhibit host-cell repair. Class II includes coliphages that contain double-stranded DNA but are assayed in repair-deficient hosts and so are not subject to host-cell repair. Class III, the least sensitive to UV inactivation, includes mammalian viruses that contain double-stranded DNA and undergo host-cell repair in competent hosts. Several conclusions can be drawn from these comparisons made on the basis of absolute dosimetry.     

Cauliflower mosaic virus: a 420 subunit (T = 7), multilayer structure.

The structures of the Cabb-B and CM1841 strains of cauliflower mosaic virus (CaMV) have been solved to about 3 nm resolution from unstained, frozen-hydrated samples that were examined with low-irradiation cryo-electron microscopy and three-dimensional image reconstruction procedures. CaMV is highly susceptible to distortions. Spherical particles, with a maximum diameter of 53.8 nm, are composed of three concentric layers (I-III) of solvent-excluded density that surround a large, solvent-filled cavity (approximately 27 nm dia). The outermost layer (I) contains 72 capsomeric morphological units, with 12 pentavalent pentamers and 60 hexavalent hexamers for a total of 420 subunits (37-42 kDa each) arranged with T = 7 icosahedral symmetry. CaMV is the first example of a T = 7 virus that obeys the rules of stoichiometry proposed for isometric viruses by Caspar and Klug (1962, Cold Spring Harb. Symp. Quant. Biol. 27, 1-24), although the hexameric capsomers exhibit marked departure from the regular sixfold symmetry expected for a structure in which the capsid protein subunits are quasi-equivalently related. The double-stranded DNA genome is distributed in layers II and III along with a portion of the viral protein. The CaMV reconstructions are consistent with the model based on neutron diffraction studies (Kruse et al., 1987, Virology 159, 166-168) and, together, these structural models are discussed in relation to a replication-assembly model (Hull et al., 1987, J. Cell Sci. (Suppl.) 7, 213-229). Remarkable agreement between the reconstructions of CaMV Cabb-B and CM1841 suggests that other strains of CaMV adopt the same basic structure.     

Molecular analysis of mutations induced by the intercalating agent ellipticine at the hisD3052 allele of Salmonella typhimurium TA98.

We have used DNA colony hybridization, the polymerase chain reaction (PCR), and direct DNA sequencing to determine the mutations induced by the intercalating agent ellipticine in Salmonella typhimurium TA98 in the presence of S9. Of 400 ellipticine-induced revertants that were selected at a mutant yield that was ninefold over the background, 85.5% contained a GC or CG deletion within a common CGCGCGCG hotspot; this deletion occurred among 47% of the spontaneous revertants. In addition to this hotspot, the ellipticine spectrum contained two deletion warmspots that reside opposite each other in looped-out regions of a possible DNA secondary structure. Ellipticine and its metabolites likely revert Salmonella strain TA98 by forming DNA adducts that promote slippage-mismatches and by stabilizing these slipped mismatched sequences via intercalation. The involvement of these mechanisms, along with a likely role for DNA secondary structures and a possible role for DNA gyrase, may account for the site specificity exhibited by ellipticine in strain TA98.     

Cellular inactivation and chromosomal aberrations: initial damage.

It has been proposed that unrepaired or misrepaired complex lesions of DNA are responsible for cell inactivation and chromosomal aberrations. The detailed features of the critical damage and the nature of initiating physical events are actively investigated. We studied the role of inner-shell (core) ionizations in DNA atoms is studied. Ultrasoft X-rays from LURE synchrotron radiation have been used to mimic core events induced by ionizing radiations. For biological matter, inner-shell photoionization is indeed the main interaction channel of these radiations. Moreover, by tuning the X-ray energy below and above the carbon K-threshold, it is possible to achieve a two-fold increase in the number of core-ionizations in DNA for a same dose. Cell survival and chromosome aberrations have thus been studied at three iso-attenuated energies: 250, 350, and 810 eV. Relative biological efficiencies (RBEs) for cell inactivation and chromosome aberrations were found to be strongly correlated with the yields of core events in DNA.     

A particle track-repeating algorithm for proton beam dose calculation

A particle track-repeating algorithm has been developed for proton beam dose calculation for radiotherapy. Monoenergetic protons with 250 MeV kinetic energy were simulated in an infinite water phantom using the GEANT3 Monte Carlo code. The changes in location, angle and energy for every transport step and the energy deposition along the track were recorded for the primary protons and all secondary particles. When calculating dose for a patient with a realistic proton beam, the pre-generated particle tracks were repeated in the patient geometry consisting of air, soft tissue and bone. The medium and density for each dose scoring voxel in the patient geometry were derived from patient CT data. The starting point, at which a proton track was repeated, was determined according to the incident proton energy. Thus, any protons with kinetic energy less than 250 MeV can be simulated. Based on the direction of the incident proton, the tracks were first rotated and for the subsequent steps, the scattering angles were simply repeated for air and soft tissue but adjusted properly based on the scattering power for bone. The particle step lengths were adjusted based on the density for air and soft tissue and also on the stopping powers for bone while keeping the energy deposition unchanged in each step. The difference in nuclear interactions and secondary particle generation between water and these materials was ignored. The algorithm has been validated by comparing the dose distributions in uniform water and layered heterogeneous phantoms with those calculated using the GEANT3 code for 120, 150, 180 and 250 MeV proton beams. The differences between them were within 2%. The new algorithm was about 13 times faster than the GEANT3 Monte Carlo code for a uniform phantom geometry and over 700 times faster for a heterogeneous phantom geometry.     

The quality of ionising radiations emitted by radionuclides incorporated into mammalian cells

Radionuclides which decay by electron capture accompanied by Auger electron cascades, or by beta emission are thought to be excessively damaging when incorporated into the molecular structure of mammalian cell nuclei and thereby pose a radiation hazard which is not assessable by conventional dosimetry. Survival data, from the literature, for 125I, 77Br, 3H and 131I have been re-analysed to extract cross sections for inactivation, by the slowing down charged particle fluence, as these are absolute specifications of the radiation quality. When comparison is made with results similarly expressed for external irradiation with heavy particle and photon beams, the qualities of the low-energy emitters 125I, 77Br and 3H are found to approach those for heavy particles. An explanation for the damage mechanism is sought in a recently developed model for radiation action. The results are consistent with the interpretation that electron damage is caused predominantly at the end of the tracks, and the actual incorporation simply ensures that the slowing down fluence of low-energy tracks interacts preferentially in the vicinity of the radiosensitive sites. The absolute biological effectiveness of these radiations can be expressed quantitatively in terms of the model parameters.     

Novel virus-like particles containing circular single-stranded DNAs associated with subterranean clover stunt disease

Novel virus-like particles, 17-19 nm in diameter, have been isolated from subterranean clover and pea plants infected with the pathogen of subterranean clover stunt disease (SCSD). The structure and genetic organization of these particles suggest that the pathogen of SCSD is representative of a new group of plant DNA viruses. SCS virus-like particles (SCSV) are isometric and band as a single component with buoyant densities of 1.24 g/ml in Cs2SO4 and 1.34 g/ml in CsCl. The A260 nm/A280 nm is about 1.35, which is consistent with an estimated nucleic acid content of 17%. Molecular calculations suggest that the particles have a T = 1 capsid structure containing 60 polypeptide subunits each with Mr of 19,000. Nucleic acid analysis including restriction enzyme digestions of double-stranded cDNAs suggests that SCSV have a divided genome composed of multiple species of circular, single-stranded DNA molecules each of approximately 850-880 nucleotides and that each is encapsidated in a separate particle. Linear and aggregated forms of these DNAs are also detected by gel electrophoresis. Evidence suggests that these virus-like particles are the pathogen of SCSD.     

The halting arrival of left-handed Z-DNA

Forty-nine years ago Watson and Crick proposed a double-stranded (ds-) model for DNA. This double helix has become an icon of molecular biology. Twenty-six years later, Rich accidently discovered Z-DNA, an exotic left-handed nucleic acid. For many years thereafter, this left-handed DNA was thought to be an artifact. DNA is no longer looked upon as a static molecule but rather an extremely dynamic structure in which different conformations are in equilibrium with each other. Many researchers have spent the last two decades characterizing this novel left-handed DNA structure. Now many investigators are beginning to accept the possibility that this novel ds-DNA conformation may play a significant in vivo role within eukaryotic and prokaryotic cells. However, more research needs to be performed before it is absolutely accepted by all in the scientific community.     

Cross-linked double-stranded RNA from a defective vesicular stomatitis virus particle

A class of vesicular stomatitis defective interfering virus particles isolated in this laboratory contains mostly double-stranded RNA of approximately 0.7–0.9 × 10⁶ daltons molecular weight. This RNA “snaps-back” or reanneals independently of concentration after heat denaturation but remains denatured if first nicked with ribonuclease. The nicked denatured products are about half the size of the double-stranded RNA and approximately half of them anneal to infectious virion single-stranded RNA. It is proposed that the complementary strands of this double-stranded RNA are cross-linked to each other, probably covalently. The structure can be viewed best as a hairpin or a closed circle base-paired along most of its length.     

In vitro assembly of a prohead-like structure of the Rhodobacter capsulatus gene transfer agent

The gene transfer agent (GTA) is a phage-like particle capable of exchanging double-stranded DNA fragments between cells of the photosynthetic bacterium Rhodobacter capsulatus. Here we show that the major capsid protein of GTA, expressed in E. coli, can be assembled into prohead-like structures in the presence of calcium ions in vitro. Transmission electron microscopy (TEM) of uranyl acetate staining material and thin sections of glutaraldehyde-fixed material demonstrates that these associates have spherical structures with diameters in the range of 27-35 nm. The analysis of scanning TEM images revealed particles of mass approximately 4.3 MDa, representing 101+/-11 copies of the monomeric subunit. The establishment of this simple and rapid method to form prohead-like particles permits the GTA system to be used for genome manipulation within the photosynthetic bacterium, for specific targeted drug delivery, and for the construction of biologically based distributed autonomous sensors for environmental monitoring.     

Psoralen preparation of antigenically intact noninfectious rotavirus particles.

The use of the synthetic psoralen 4'-aminomethyl-4,5',8-trimethylpsoralen hydrochloride (AMT) is described for the inactivation of infectious rotavirus, a member of the viral family Reoviradae with a double-stranded RNA genome. This method not only provides complete inactivation of the virus but leaves antigenically intact particles. The lack of viral replication following inactivation was determined with an immunohistochemical focus assay. The antigenic authenticity of the particles was determined by monoclonal antibody ELISA and a viral hemagglutination assay.     

Measurement and analysis of supralinearity in LiF TLD-100 irradiated by 1.5 keV x-rays

Measurements of the response of lithium fluoride TLD-100 to 1.5 keV ultrasoft x-rays, 240 keV x-rays and 137Cs gamma-rays at doses below 70 Gy are presented and the implications for the track interaction model are discussed. This model proposes that thermoluminescence is due to the combination electrons and holes formed along secondary charged particle tracks. As the dose is increased, the average distance between particle tracks is reduced and there is a greater probability of combination between neighbouring tracks; this mechanism is used to explain the supralinear response of lithium fluoride to certain radiations. Measurements using ultrasoft x-rays are interesting in this context because the energy from each photon is deposited in a small volume, therefore the probability of combination between neighbouring events should only be significant at high doses. The results, however, show that lithium fluoride exhibits a significant supralinear response to 1.5 keV x-rays after only a few tens of grays and suggests that the track interaction model alone cannot explain the phenomenon of supralinearity.     

Communication between Hin recombinase and Fis regulatory subunits during coordinate activation of Hin-catalyzed site-specific DNA inversion

The Hin DNA invertase becomes catalytically activated when assembled in an invertasome complex containing two Fis dimers bound to an enhancer segment. The region of Fis responsible for transactivation of Hin contains a mobile β-hairpin arm that extends from each dimer subunit. We show here that whereas both Fis dimers must be capable of activating Hin, Fis heterodimers that have only one functional activating β-arm are sufficient to form catalytically competent invertasomes. Analysis of homodimer and heterodimer mixes of different Hin mutants suggests that Fis must activate each subunit of the two Hin dimers that participate in catalysis. These experiments also indicate that all four Hin subunits must be coordinately activated prior to initiation of the first chemical step of the reaction and that the process of activation is independent of the catalytic steps of recombination. We propose a molecular model for the invertasome structure that is consistent with current information on protein–DNA structures and the topology of the DNA strands within the recombination complex. In this model, a single Fis activation arm could contact amino acids from both Hin subunits at the dimer interface to induce a conformational change that coordinately positions the active sites close to the scissile phosphodiester bonds.