TOPAS-nBio validation for simulating water radiolysis and DNA damage under low-LET irradiation.

The chemical stage of the Monte Carlo track-structure simulation code Geant4-DNA has been revised and validated. The root-mean-square (RMS) empirical parameter that dictates the displacement of water molecules after an ionization and excitation event in Geant4-DNA has been shortened to better fit experimental data. The pre-defined dissociation channels and branching ratios were not modified, but the reaction rate coefficients for simulating the chemical stage of water radiolysis were updated. The evaluation of Geant4-DNA was accomplished with TOPAS-nBio. For that, we compared predicted time-dependentGvalues in pure liquid water for·OH, e-aq, and H2with published experimental data. For H2O2and H·, simulation of added scavengers at different concentrations resulted in better agreement with measurements. In addition, DNA geometry information was integrated with chemistry simulation in TOPAS-nBio to realize reactions between radiolytic chemical species and DNA. This was used in the estimation of the yield of single-strand breaks (SSB) induced by137Csγ-ray radiolysis of supercoiled pUC18 plasmids dissolved in aerated solutions containing DMSO. The efficiency of SSB induction by reaction between radiolytic species and DNA used in the simulation was chosen to provide the best agreement with published measurements. An RMS displacement of 1.24 nm provided agreement with measured data within experimental uncertainties for time-dependentGvalues and under the presence of scavengers. SSB efficiencies of 24% and 0.5% for·OH and H·, respectively, led to an overall agreement of TOPAS-nBio results within experimental uncertainties. The efficiencies obtained agreed with values obtained with published non-homogeneous kinetic model and step-by-step Monte Carlo simulations but disagreed by 12% with published direct measurements. Improvement of the spatial resolution of the DNA damage model might mitigate such disagreement. In conclusion, with these improvements, Geant4-DNA/TOPAS-nBio provides a fast, accurate, and user-friendly tool for simulating DNA damage under low linear energy transfer irradiation.

Intermittent compression of the calf muscle as a countermeasure to protect blood pressure and brain blood flow in upright posture in older adults.

PURPOSE: Orthostatic hypotension, leading to cerebral hypoperfusion, can result in postural instability and falls in older adults. We determined the efficacy of a novel, intermittent pneumatic compression system, applying pressure around the lower legs, as a countermeasure against orthostatic stress in older adults. METHODS: Data were collected from 13 adults (4 male) over 65 years of age. Non-invasive ultrasound measured middle cerebral artery blood velocity (MCAv) and finger photoplethysmography measured mean arterial blood pressure (MAP). Intermittent lower leg compression was applied in a peristaltic manner in the local diastolic phase of each cardiac cycle to optimize venous return during 1-min of seated rest and during a sit-to-stand transition to 1-min of quiet standing with compression initiated 15 s before transition. RESULTS: During seated rest, compression resulted in a 4.5 ± 6.5 mmHg increase in MAP, and 2.3 ± 2.1 cm/s increase in MCAv (p < 0.05). MAP and MCAv increased during the 15 s of applied compression before the posture transition (2.3 ± 7.2 mmHg and 2.1 ± 4.0 cm/s, respectively, p < 0.05) with main effects for both variables confirming continued benefit during the transition and quiet stand periods. CONCLUSIONS: Application of carefully timed, intermittent compression to the lower legs of older adults increased MAP and MCAv during seated rest and maintained an elevated MAP and MCAv during a transition to standing posture. Future research could assess the benefits of this technology for persons at risk for orthostatic hypotension on standing and while walking in an effort to reduce injurious, unexplained falls in older adults.

Bio-based Algae Oil: an oxidation and structural analysis.

OBJECTIVE: In search of natural components, vegetal oils are increasingly becoming more popular in cosmetics. However, high oxidation instability, presence of potential allergens and synthetic anti-oxidants have limited their applications so far. Therefore, a need exists for a natural emollient with high oxidation stability. In this work, we report on a novel sustainably produced triglyceride containing primarily three monounsaturated oleic acid chains, dubbed 'Bio-Based Algae Oil' hereafter, as a natural emollient for cosmetic formulations. To produce Bio-Based Algae Oil, simple sugars are converted into triglyceride oils using microalgae fermentation with minimal environmental impact. METHODS: Bio-Based Algae Oil was compared to other commonly used triglyceride-based emollients in the skincare industry in terms of thermal/oxidation stability, composition and moisturizing properties. Oxidation stability of emollients was compared using Rancimat and pressurized differential scanning calorimetry (PDSC) techniques. Fatty acid composition of each oil was analysed using proton nuclear magnetic resonance (1 H-NMR) and gas chromatography (GC) techniques to correlate unsaturation level of each oil to its oxidation stability. We also conducted an in vivo moisturizing study in which skin hydration level of human subjects was compared before and after application of emollient up to 24 h. RESULTS: Results showed that Bio-Based Algae Oil was the most stable emollient in thermal and oxidation stability studies given its low unsaturation and high anti-oxidant content determined by 1 H-NMR and GC techniques. It also provided the highest skin hydration level when applied on skin demonstrating its efficacy as a moisturizing emollient in cosmetic formulations. CONCLUSIONS: Compositional analysis of Bio-Based Algae revealed that it is a triglyceride containing primarily three monounsaturated oleic acid chains with very low polyunsaturated fatty acid content resulting in high oxidation stability and consequently prolonged shelf-life. Given its sustainability, high oxidation stability and skin health benefits such as moisturization demonstrated during an in vivo study, we envision to utilize Bio-Based Algae Oil in many cosmetic formulations across skincare, suncare and bath and shower markets.

OBJECTIF: Dans la quête de composants naturels, les huiles végétales deviennent de plus en plus prisées en cosmétique. Toutefois, la forte instabilité à l'oxydation, la présence d'allergènes potentiels et d'antioxydants synthétiques ont contribué à la réduction de leurs demandes jusqu'ici. Par conséquent, il existe un besoin en émollient naturel doté d'une stabilité à l'oxydation élevée. Dans le cadre de ces travaux, nous présentons un nouveau triglycéride produit de manière durable contenant principalement trois chaînes d'acide oléique mono-insaturées, appelé « Huile d'algues d'origine biologique ¼ comme étant un émollient naturel pour les produits cosmétiques. Pour obtenir de l'Huile d'algues d'origine biologique, des sucres rapides sont transformés en huiles triglycérides par fermentation de microalgues avec un impact environnemental minimal. MÉTHODES: L'huile d'algues d'origine biologique a été comparée à d'autres émollients à base de triglycérides fréquemment utilisés dans l'industrie des soins de la peau en matière de stabilité thermique ou d'oxydation, de composition et de propriétés hydratantes. Stabilité thermique ou oxydation, composition et propriétés hydratantes. La stabilité à l'oxydation des émollients a été comparée grâce au Rancimat et à des techniques de calorimétrie différentielle à balayage haute pression (PDSC). La composition des acides gras de chaque huile a été analysée grâce aux techniques de résonance magnétique nucléaire du proton (1 H-NMR) et de chromatographie en phase gazeuse (CPG) afin de créer une corrélation entre le taux d'insaturation de chaque huile et sa stabilité à l'oxydation. Nous avons également procédé à une étude in vivo de l'hydratation au cours de laquelle le niveau d'hydratation de la peau des patients humains a été comparé avant et après l'utilisation de l'émollient sur une période allant jusqu'à 24 h. RÉSULTATS: Les résultats ont démontré que l'huile d'algues d'origine biologique était l'émollient le plus stable lors des études sur la stabilité thermique et la stabilité à l'oxydation, en raison de sa faible insaturation et de sa teneur élevée en antioxydants déterminés par les techniques 1 H-NMR et GC. Elle a également produit le taux d'hydratation de la peau le plus élévé, lorsqu'appliqué à la peau, ce qui démontre son efficacité comme émollient hydratant dans les produits cosmétiques. CONCLUSIONS: L'analyse de la composition de l'huile d'algues d'origine biologique a révélé qu'il s'agit d'un triglycéride contenant principalement trois chaînes d'acide oléique mono-insaturées avec une très faible teneur en acides gras polyinsaturés, ce qui entraîne une stabilité à l'oxydation élevée et par conséquent une durée de vie prolongée. Compte tenu de sa durabilité, sa stabilité à l'oxydation élevée et ses bienfaits pour la santé de la peau, notamment l'hydratation démontrée au cours d'une étude in vivo, nous envisageons d'utiliser l'huile d'algues d'origine biologique dans de nombreuses formulations cosmétiques présentes sur le marché des soins de la peau, des produits solaires et des bains et douches.

Comparison of portable blood-warming devices under simulated pre-hospital conditions: a randomised in-vitro blood circuit study.

Pre-hospital transfusion of blood products is a vital component of many advanced pre-hospital systems. Portable fluid warmers may be utilised to help prevent hypothermia, but the limits defined by manufacturers often do not reflect their clinical use. The primary aim of this randomised in-vitro study was to assess the warming performance of four portable blood warming devices (Thermal Angel, Hypotherm X LG, °M Warmer, Buddy Lite) against control at different clinically-relevant flow rates. The secondary aim was to assess haemolysis rates between devices at different flow rates. We assessed each of the four devices and the control, at flow rates of 50 ml.min-1 , 100 ml.min-1 and 200 ml.min-1 , using a controlled perfusion circuit with multisite temperature monitoring. Free haemoglobin concentration, a marker of haemolysis, was measured at multiple points during each initial study run with spectrophotometry. At all flow rates, the four devices provided superior warming performance compared with the control (p < 0.001). Only the °M Warmer provided a substantial change in temperature at all flow rates (mean (95%CI) temperature change of 21.1 (19.8-22.4) °C, 20.4 (19.1-21.8) °C and 19.4 (17.7-21.1) °C at 50 ml.min-1 , 100 ml.min-1 and 200 ml.min-1 , respectively). There was no association between warming and haemolysis with any device (p = 0.949) or flow rate (p = 0.169). Practical issues, which may be relevant to clinical use, also emerged during testing. Our results suggest that there were significant differences in the performance of portable blood warming devices used at flow rates encountered in clinical practice.

A New Standard DNA Damage (SDD) Data Format.

Our understanding of radiation-induced cellular damage has greatly improved over the past few decades. Despite this progress, there are still many obstacles to fully understand how radiation interacts with biologically relevant cellular components, such as DNA, to cause observable end points such as cell killing. Damage in DNA is identified as a major route of cell killing. One hurdle when modeling biological effects is the difficulty in directly comparing results generated by members of different research groups. Multiple Monte Carlo codes have been developed to simulate damage induction at the DNA scale, while at the same time various groups have developed models that describe DNA repair processes with varying levels of detail. These repair models are intrinsically linked to the damage model employed in their development, making it difficult to disentangle systematic effects in either part of the modeling chain. These modeling chains typically consist of track-structure Monte Carlo simulations of the physical interactions creating direct damages to DNA, followed by simulations of the production and initial reactions of chemical species causing so-called "indirect" damages. After the induction of DNA damage, DNA repair models combine the simulated damage patterns with biological models to determine the biological consequences of the damage. To date, the effect of the environment, such as molecular oxygen (normoxic vs. hypoxic), has been poorly considered. We propose a new standard DNA damage (SDD) data format to unify the interface between the simulation of damage induction in DNA and the biological modeling of DNA repair processes, and introduce the effect of the environment (molecular oxygen or other compounds) as a flexible parameter. Such a standard greatly facilitates inter-model comparisons, providing an ideal environment to tease out model assumptions and identify persistent, underlying mechanisms. Through inter-model comparisons, this unified standard has the potential to greatly advance our understanding of the underlying mechanisms of radiation-induced DNA damage and the resulting observable biological effects when radiation parameters and/or environmental conditions change.

Using RecA protein to enhance kinetic rates of DNA circuits.

While DNA circuits are becoming increasingly useful as signal transducers, their utility is inhibited by their slow catalytic rate. Here, we demonstrate how RecA, a recombination enzyme that catalyzes sequence specific strand exchange, can be used to increase circuit rates up to 9-fold. We also show how the introduction of RNA into DNA circuits further controls the specificity of RecA strand exchange, improving signal-to-noise.

Substandard and falsified anti-tuberculosis drugs: a preliminary field analysis.

SETTING: Pharmacies in 19 cities in Angola, Brazil, China, Democratic Republic of Congo, Egypt, Ethiopia, Ghana, India (n = 3), Kenya, Nigeria, Russia, Rwanda, Thailand, Turkey, Uganda, United Republic of Tanzania and Zambia. OBJECTIVE: To assess the quality of the two main first-line anti-tuberculosis medicines, isoniazid and rifampicin, procured from private-sector pharmacies, to determine if substandard and falsified medicines are available and if they potentially contribute to drug resistance in cities in low- and middle-income countries. DESIGN: Local nationals procured 713 treatment packs from a selection of pharmacies in 19 cities. These samples were tested for quality using 1) thin-layer chromatography to analyze levels of active pharmaceutical ingredient (API), and 2) disintegration testing. RESULTS: Of 713 samples tested, 9.1% failed basic quality testing for requisite levels of API or disintegration. The failure rate was 16.6% in Africa, 10.1% in India, and 3.9% in other middle-income countries. CONCLUSIONS: Substandard and falsified drugs are readily available in the private marketplace and probably contribute to anti-tuberculosis drug resistance in low- and middle-income countries. This issue warrants further investigation through large-scale studies of drug quality in all markets.

Spontaneous debulking of middle fossa chordoma extension after transnasal petroclival biopsy--report of a case.

BACKGROUND: Clival chordomas are difficult tumors to treat, particularly when they have already grown beyond the confines of the clivus. PATIENT: We report the case of a 52-year-old man with a clival mass consistent with a chordoma with a prominent extension into the right middle fossa. At the patient's request, he underwent a simple endonasal biopsy to confirm the diagnosis. A second debulking procedure was planned to debulk the remnant tumor. However, follow-up magnetic resonance imaging showed that much of the middle fossa tumor had decompressed itself through the clival defect into the patient's pharynx. RESULTS: The patient underwent additional clival debulking and proton-beam therapy. After 44 months of follow-up, he had no clinical or radiographic progression of disease. CONCLUSION: It is intriguing to think that leaving a path for easy egress for a chordoma from the clivus may prevent it from building up in the bone and spreading.

How can we improve our understanding of cardiovascular safety liabilities to develop safer medicines?

Given that cardiovascular safety liabilities remain a major cause of drug attrition during preclinical and clinical development, adverse drug reactions, and post-approval withdrawal of medicines, the Medical Research Council Centre for Drug Safety Science hosted a workshop to discuss current challenges in determining, understanding and addressing 'Cardiovascular Toxicity of Medicines'. This article summarizes the key discussions from the workshop that aimed to address three major questions: (i) what are the key cardiovascular safety liabilities in drug discovery, drug development and clinical practice? (ii) how good are preclinical and clinical strategies for detecting cardiovascular liabilities? and (iii) do we have a mechanistic understanding of these liabilities? It was concluded that in order to understand, address and ultimately reduce cardiovascular safety liabilities of new therapeutic agents there is an urgent need to: • Fully characterize the incidence, prevalence and impact of drug-induced cardiovascular issues at all stages of the drug development process. • Ascertain the predictive value of existing non-clinical models and assays towards the clinical outcome. • Understand the mechanistic basis of cardiovascular liabilities; by addressing areas where it is currently not possible to predict clinical outcome based on preclinical safety data. • Provide scientists in all disciplines with additional skills to enable them to better integrate preclinical and clinical data and to better understand the biological and clinical significance of observed changes. • Develop more appropriate, highly relevant and predictive tools and assays to identify and wherever feasible to eliminate cardiovascular safety liabilities from molecules and wherever appropriate to develop clinically relevant and reliable safety biomarkers.

A nanodosimetric model of radiation-induced clustered DNA damage yields.

We present a nanodosimetric model for predicting the yield of double strand breaks (DSBs) and non-DSB clustered damages induced in irradiated DNA. The model uses experimental ionization cluster size distributions measured in a gas model by an ion counting nanodosimeter or, alternatively, distributions simulated by a Monte Carlo track structure code developed to simulate the nanodosimeter. The model is based on a straightforward combinatorial approach translating ionizations, as measured or simulated in a sensitive gas volume, to lesions in a DNA segment of one-two helical turns considered equivalent to the sensitive volume of the nanodosimeter. The two model parameters, corresponding to the probability that a single ion detected by the nanodosimeter corresponds to a single strand break or a single lesion (strand break or base damage) in the equivalent DNA segment, were tuned by fitting the model-predicted yields to previously measured double-strand break and double-strand lesion yields in plasmid DNA irradiated with protons and helium nuclei. Model predictions were also compared to both yield data simulated by the PARTRAC code for protons of a wide range of different energies and experimental DSB and non-DSB clustered DNA damage yield data from the literature. The applicability and limitations of this model in predicting the LET dependence of clustered DNA damage yields are discussed.

Kinetic behavior of the reaction between hydroxyl radical and the SV40 minichromosome.

Aqueous solutions containing the minichromosomal form of the virus SV40 and the radical scavenger DMSO were subjected to gamma-irradiation, and the resulting formation of single strand breaks (SSB) was quantified. Under the irradiation conditions, most SSBs were produced as a consequence of hydroxyl radical ((•)OH) reactions. By controlling the competition between DMSO and the viral DNA substrate for (•)OH, we are able to estimate the rate coefficient for the reaction of (•)OH with the SV40 minichromosome. The results cannot be described adequately by homogeneous competition kinetics, but it is possible to describe the rate coefficient for the reaction as a function of the scavenging capacity of the solution. The experimentally determined rate coefficient lies in the range 1×10(9) - 2×10(9) L mol(-1) s(-1) at 10(7) s(-1), and increases with increasing scavenging capacity.

In-vitro evaluation of the PALL Leukotrap Affinity Prion Reduction Filter as a secondary device following primary leucoreduction.

BACKGROUND AND OBJECTIVES: A filter (PRF1) designed to remove abnormal prion proteins from red-cell units has been developed. The purpose of this study was to evaluate the quality of red cells produced using this device. MATERIALS AND METHODS: Leucocyte-depleted red-cell units (CPD, CPD-A1 and CPD/SAGM) processed according to standard UK practices were filtered using PRF1. Filtered and control units were stored and sampled on day 1, day 7 and on the date of expiry and were tested using standard measures of red-cell quality. RESULTS: Filtered units were found to have significantly higher percentage haemolysis levels, lower haemoglobin levels and a smaller volume compared with controls. All results, however, were well within the permitted 0.8% haemolysis level at the end of storage and all units met the UK guidelines for haemoglobin and volume. The other test parameters measured showed no significant differences between the test and control units. CONCLUSIONS: The PRF1 filter was found to be easy to use and resulted in red-cell units that met all relevant UK and European Guidelines.

Evaluation of lesion clustering in irradiated plasmid DNA.

PURPOSE: To measure the yield of DNA strand breaks and clustered lesions in plasmid DNA irradiated with protons, helium nuclei, and y-rays. MATERIALS AND METHODS: Plasmid DNA was irradiated with 1.03, 19.3 and 249 MeV protons (linear energy transfer = 25.5, 2.7, and 0.39 keV microm(-1) respectively), 26 MeV helium nuclei (25.5 keV microm) and gamma-rays (137Cs or 60Co) in phosphate buffer containing 2 mM or 200 mM glycerol. Single-and double-strand breaks (SSB and DSB) were measured by gel electrophoresis, and clustered lesions containing base lesions were quantified by converting them into irreparable DSB in transformed bacteria. RESULTS: For protons, SSB yield decreased with increasing LET (linear energy transfer). The yield of DSB and all clustered lesions seemed to reach a minimum around 3 keV microm(-1). There was a higher yield of SSB, DSB and total clustered lesions for protons compared to helium nuclei at 25.5 keV microm(-1). A difference in the yields between 137Cs and 60Co gamma-rays was also observed, especially for SSB. CONCLUSION: In this work we have demonstrated the complex LET dependence of clustered-lesion yields, governed by interplay of the radical recombination and change in track structure. As expected, there was also a significant difference in clustered lesion yields between various radiation fields, having the same or similar LET values, but differing in nanometric track structure.

Radioprotection of plasmid DNA by oligolysines.

PURPOSE: To define ionic conditions under which oligolysines condense DNA as assayed by radioprotection of a plasmid substrate. And to compare these conditions with those required by the well-characterized ligands spermidine and hexammine cobalt (III). This will enable a reversible compaction model for plasmid DNA to be devised that models more closely mammalian chromatin than those based on polyamines. MATERIALS AND METHODS: Aqueous solutions containing plasmid DNA, sodium perchlorate and one of the five ligands trilysine, tetralysine, pentalysine, spermidine, or hexammine cobalt (III) were subjected to gamma-irradiation. The yields of the resulting single-strand breaks were quantified by gel electrophoresis. The effects of tetralysine and pentalysine were also examined by light scattering. RESULTS: The combination of low concentrations of the ligand and high concentrations of sodium perchlorate produced a relatively high yield of single-strand breaks. In contrast, the combination of high concentrations of the ligand and low concentrations of sodium perchlorate resulted in an approximately 25-fold lower single-strand break yield. The transition between these two break yields took place over very narrow concentration ranges of the ligand. A large change in light scattering occurred at the same concentration. The radioprotective ability of the ligands decreased in the order pentalysine > tetralysine > hexammine cobalt (III) > spermidine > trilysine. CONCLUSIONS: The effect of the oligolysines is qualitatively very similar to the previously reported radioprotection produced under similar conditions by the polyamines spermidine and spermine. It is caused by condensation of the DNA into a highly compacted form. As peptides, oligolysines are structurally more closely related than other ligands to naturally occurring DNA condensing agents such as histone proteins. Therefore, they may form the basis of a model system suitable for studying DNA damage produced by the direct effect of ionizing radiation (ionization of the DNA itself).

Immune cell regulation and cardiovascular effects of sphingosine 1-phosphate receptor agonists in rodents are mediated via distinct receptor subtypes.

Sphingosine 1-phosphate (S1P) is a bioactive lysolipid with pleiotropic functions mediated through a family of G protein-coupled receptors, S1P(1,2,3,4,5). Physiological effects of S1P receptor agonists include regulation of cardiovascular function and immunosuppression via redistribution of lymphocytes from blood to secondary lymphoid organs. The phosphorylated metabolite of the immunosuppressant agent FTY720 (2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol) and other phosphonate analogs with differential receptor selectivity were investigated. No significant species differences in compound potency or rank order of activity on receptors cloned from human, murine, and rat sources were observed. All synthetic analogs were high-affinity agonists on S1P(1), with IC(50) values for ligand binding between 0.3 and 14 nM. The correlation between S1P(1) receptor activation and the ED(50) for lymphocyte reduction was highly significant (p < 0.001) and lower for the other receptors. In contrast to S1P(1)-mediated effects on lymphocyte recirculation, three lines of evidence link S1P(3) receptor activity with acute toxicity and cardiovascular regulation: compound potency on S1P(3) correlated with toxicity and bradycardia; the shift in potency of phosphorylated-FTY720 for inducing lymphopenia versus bradycardia and hypertension was consistent with affinity for S1P(1) relative to S1P(3); and toxicity, bradycardia, and hypertension were absent in S1P(3)(-/-) mice. Blood pressure effects of agonists in anesthetized rats were complex, whereas hypertension was the predominant effect in conscious rats and mice. Immunolocalization of S1P(3) in rodent heart revealed abundant expression on myocytes and perivascular smooth muscle cells consistent with regulation of bradycardia and hypertension, whereas S1P(1) expression was restricted to the vascular endothelium.

Repair of oxidative DNA damage by amino acids.

Guanyl radicals, the product of the removal of a single electron from guanine, are produced in DNA by the direct effect of ionizing radiation. We have produced guanyl radicals in DNA by using the single electron oxidizing agent (SCN)2-, itself derived from the indirect effect of ionizing radiation via thiocyanate scavenging of OH. We have examined the reactivity of guanyl radicals in plasmid DNA with the six most easily oxidized amino acids cysteine, cystine, histidine, methionine, tryptophan and tyrosine and also simple ester and amide derivatives of them. Cystine and histidine derivatives are unreactive. Cysteine, methionine, tyrosine and particularly tryptophan derivatives react to repair guanyl radicals in plasmid DNA with rate constants in the region of approximately 10(5), 10(5), 10(6) and 10(7) dm3 mol(-1) s(-1), respectively. The implication is that amino acid residues in DNA binding proteins such as histones might be able to repair by an electron transfer reaction the DNA damage produced by the direct effect of ionizing radiation or by other oxidative insults.

Ion-counting nanodosimetry: current status and future applications.

There is a growing interest in the study of interactions of ionizing radiation with condensed matter at the nanometer level. The motivation for this research is the hypothesis that the number of ionizations occurring within short segments of DNA-size subvolumes is a major factor determining the biological effectiveness of ionizing radiation. A novel dosimetry technique, called nanodosimetry, measures the spatial distribution of individual ionizations in an irradiated low-pressure gas model of DNA. The measurement of nanodosimetric event size spectra may enable improved characterization of radiation quality, with applications in proton and charged-particle therapy, radiation protection, and space research. We describe an ion-counting nanodosimeter developed for measuring radiation-induced ionization clusters in small, wall-less low-pressure gas volumes, simulating short DNA segments. It measures individual radiation-induced ions, deposited in 1 Torr propane within a tissue-equivalent cylindrical volume of 2-4 nm diameter and up to 100 nm length. We present first ionization cluster size distributions obtained with 13.6 MeV protons, 4.25 MeV alpha particles and 24.8 MeV carbon nuclei in propane; they correspond to a wide LET range of 4-500 keV/microm. We are currently developing plasmid-based assays to characterize the local clustering of DNA damage with biological methods. First results demonstrate that there is increasing complexity of DNA damage with increasing LET. Systematic comparison of biological and nanodosimetric data will help us to validate biophysical models predicting radiation quality based on nanodosimetric spectra. Possible applications for charged particle radiation therapy planning are discussed.

Modification of ionizing radiation clustered damage: estimate of the migration distance of holes through DNA via guanyl radicals under physiological conditions.

PURPOSE: Guanyl radicals are produced in DNA when it is subjected to oxidation or ionizing radiation. The sites at which stable products can be identified can be located dozens of base pairs away from the initial site of the electron loss. This migration will modify the spatial distribution of damage and tends to mitigate the clustering of initial damage generally associated with ionizing radiation. The migration distance is presumably a function of the lifetime of the intermediate guanyl radical, and we wished to quantify the relationship between them. MATERIALS AND METHODS: Aqueous solutions containing plasmid DNA and thiocyanate ions were treated with gamma-irradiation. These conditions result in the very efficient production of guanyl radicals in the plasmid. We quantified the formation of stable guanine oxidation products in the plasmid as strand breaks by using the E. coli base excision repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG). The effect of two additives on the yield of guanine oxidation, nitrite ions and the DNA binding ligand doxorubicin (adriamycin), were examined. RESULTS: The presence during irradiation of the DNA-binding ligand doxorubicin attenuated the yields of stable oxidized guanine products formed. The additional presence of nitrite decreased this effect of doxorubicin. CONCLUSION: Because doxorubicin binds strongly to DNA, its ability to attenuate guanine oxidation can be interpreted in terms of the migration distance of the intermediate guanyl radical. Because nitrite repairs these intermediate guanyl radicals by electron transfer, its presence during irradiation decreases their lifetime. Therefore, we derived an estimate of the migration distance of guanyl radicals as a function of their lifetime. The presence in cells of antioxidants such as glutathione sets an upper limit to the likely lifetime and, therefore, the migration distance of guanyl radicals. It was concluded that the migration of guanyl radicals may not decrease the clustering of DNA damage in vivo to a great extent.

Bistranded oxidized purine damage clusters: induced in DNA by long-wavelength ultraviolet (290-400 nm) radiation?

Bistranded clustered DNA damages involving oxidized bases, abasic sites, and strand breaks are produced by ionizing radiation and radiomimetic drugs, but it was not known whether they can be formed by other agents, e.g., nonionizing radiation. UV radiation produces clusters of cyclobutyl pyrimidine dimers, photoproducts that occur individually in high yield. Since long-wavelength UV (290-400 nm) radiation induces oxidized bases, abasic sites, and strand breaks at low yields, we tested whether it also produces clusters containing these lesions. We exposed supercoiled pUC18 DNA to UV radiation with wavelengths of >290 nm (UVB plus UVA radiation), and assessed the induction of bistranded clustered oxidized purine and abasic clusters, as recognized by Escherichia coli Fpg protein and E. coli Nfo protein (endonuclease IV), respectively, as well as double-strand breaks. These three classes of bistranded clusters were detected, albeit at very low yields (37 Fpg-OxyPurine clusters Gbp(-1) kJ(-1) m(2), 8.1 double-strand breaks Gbp(-1) kJ(-1) m(2), and 3.4 Nfo-abasic clusters Gbp(-1) kJ(-1) m(2)). Thus, these bistranded OxyPurine clusters, abasic clusters, and double-strand breaks are not uniquely induced by ionizing radiation and radiomimetic drugs, but their level of production by UVB and UVA radiation is negligible compared to the levels of frequent photoproducts such as pyrimidine dimers.

One-electron oxidation of plasmid DNA by selenium(V) species.

PURPOSE: To employ the gamma-radiation-generated selenium(V) one-electron-oxidizing agent SeO3*- for the preparation of guanyl radicals in plasmid DNA, and to compare the behaviour of this reagent with that of other similarly reactive oxidant species. MATERIALS AND METHODS: Plasmid DNA in aerobic aqueous solution was irradiated with 137Cs gamma-rays (662 keV). The solutions also contained up to 4x10(-2) mol x dm(-3) sodium selenate (Na2SeO4) and/or up to 10(-1) mol x dm(-3) sodium biselenite (NaHSeO3), as well as auxiliary scavengers such as DMSO or glycerol. In some cases, reducing agents such as ferrocyanide were also present. After irradiation, the plasmid was incubated with the Escherichia coli base excision-repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG). These treatments produced strand breaks in the plasmid. The yields of these strand breaks were quantified by agarose gel electrophoresis. RESULTS: In general, gamma-irradiation produced single-strand breaks (SSB) in plasmid DNA. Subsequent incubation with the endonuclease FPG increased the SSB yield by a factor of 2-100-fold. The smallest effects of FPG were observed when only DMSO or glycerol were present during irradiation. FPG incubation produced significantly larger increases in the SSB yield after gamma-irradiation in the additional presence of selenate and/or biselenite. The largest effect of FPG was observed after gamma-irradiation in the presence of 10(-2) mol x dm(-3) sodium selenate and 10(-1) mol x dm(-3) glycerol. This was indicative of extensive oxidative damage to the plasmid under these conditions and provided evidence for guanine oxidation mediated by SeO3*-. The large effect of FPG was strongly attenuated by the addition of reducing agents such as ferrocyanide. The observations suggest that these reducing agents exert their effects through the reduction of an intermediate guanyl radical. CONCLUSION: By comparing the yields of breaks produced after gamma-irradiation under a range of conditions, it is possible to formulate a reaction scheme that describes the chemical reactions responsible for the formation of strand breaks and FPG-sensitive sites. By applying this scheme to the data, we can quantify rate constants for the reduction of DNA guanyl radicals by reducing agents. This reaction is of particular interest to radiation biology because it is the equivalent of the repair of DNA damage by the direct effect of ionizing radiation.