Modeling radiation-induced cell death: role of different levels of DNA damage clustering.

Some open questions on the mechanisms underlying radiation-induced cell death were addressed by a biophysical model, focusing on DNA damage clustering and its consequences. DNA "cluster lesions" (CLs) were assumed to produce independent chromosome fragments that, if created within a micrometer-scale threshold distance (d), can lead to chromosome aberrations following mis-rejoining; in turn, certain aberrations (dicentrics, rings and large deletions) were assumed to lead to clonogenic cell death. The CL yield and d were the only adjustable parameters. The model, implemented as a Monte Carlo code called BIophysical ANalysis of Cell death and chromosome Aberrations (BIANCA), provided simulated survival curves that were directly compared with experimental data on human and hamster cells exposed to photons, protons, α-particles and heavier ions including carbon and iron. d = 5 µm, independent of radiation quality, and CL yields in the range ~2-20 CLs Gy(-1) cell(-1), depending on particle type and energy, led to good agreement between simulations and data. This supports the hypothesis of a pivotal role of DNA cluster damage at sub-micrometric scale, modulated by chromosome fragment mis-rejoining at micrometric scale. To investigate the features of such critical damage, the CL yields were compared with experimental or theoretical yields of DNA fragments of different sizes, focusing on the base-pair scale (related to the so-called local clustering), the kbp scale ("regional clustering") and the Mbp scale, corresponding to chromatin loops. Interestingly, the CL yields showed better agreement with kbp fragments rather than bp fragments or Mbp fragments; this suggests that also regional clustering, in addition to other clustering levels, may play an important role, possibly due to its relationship with nucleosome organization in the chromatin fiber.

Boron concentration measurement in biological tissues by charged particle spectrometry.

Measurement of boron concentration in biological tissues is a fundamental aspect of boron neutron capture therapy, because the outcome of the therapy depends on the distribution of boron at a cellular level, besides on its overall concentration. This work describes a measurement technique based on the spectroscopy of the charged particles emitted in the reaction (10)B(n,α)(7)Li induced by thermal neutrons, allowing for a quantitative determination of the boron concentration in the different components that may be simultaneously present in a tissue sample, such as healthy cells, tumor cells and necrotic cells. Thin sections of tissue containing (10)B are cut at low temperatures and irradiated under vacuum in a thermal neutron field. The charged particles arising from the sample during the irradiation are collected by a thin silicon detector, and their spectrum is used to determine boron concentration through relatively easy calculations. The advantages and disadvantages of this technique are here described, and validation of the method using tissue standards with known boron concentrations is presented.

Thermal neutron irradiation field design for boron neutron capture therapy of human explanted liver.

The selective uptake of boron by tumors compared to that by healthy tissue makes boron neutron capture therapy (BNCT) an extremely advantageous technique for the treatment of tumors that affect a whole vital organ. An example is represented by colon adenocarcinoma metastases invading the liver, often resulting in a fatal outcome, even if surgical resection of the primary tumor is successful. BNCT can be performed by irradiating the explanted organ in a suitable neutron field. In the thermal column of the Triga Mark II reactor at Pavia University, a facility was created for this purpose and used for the irradiation of explanted human livers. The neutron field distribution inside the organ was studied both experimentally and by means of the Monte Carlo N-particle transport code (MCNP). The liver was modeled as a spherical segment in MCNP and a hepatic-equivalent solution was used as an experimental phantom. In the as-built facility, the ratio between maximum and minimum flux values inside the phantom ((phi(max)/phi(min)) was 3.8; this value can be lowered to 2.3 by rotating the liver during the irradiation. In this study, the authors proposed a new facility configuration to achieve a uniform thermal neutron flux distribution in the liver. They showed that a phi(max)/phi(min) ratio of 1.4 could be obtained without the need for organ rotation. Flux distributions and dose volume histograms were reported for different graphite configurations.

Dose distributions in phantoms irradiated in thermal columns of two different nuclear reactors.

In-phantom dosimetry studies have been carried out at the thermal columns of a thermal- and a fast-nuclear reactor for investigating: (a) the spatial distribution of the gamma dose and the thermal neutron fluence and (b) the accuracy at which the boron concentration should be estimated in an explanted organ of a boron neutron capture therapy patient. The phantom was a cylinder (11 cm in diameter and 12 cm in height) of tissue-equivalent gel. Dose images were acquired with gel dosemeters across the axial section of the phantom. The thermal neutron fluence rate was measured with activation foils in a few positions of this phantom. Dose and fluence rate profiles were also calculated with Monte Carlo simulations. The trend of these profiles do not show significant differences for the thermal columns considered in this work.

The role of DNA cluster damage and chromosome aberrations in radiation-induced cell killing: a theoretical approach.

The role played by DNA cluster damage and chromosome aberrations in radiation-induced cell killing was investigated, assuming that certain chromosome aberrations (dicentrics, rings and large deletions, or 'lethal aberrations') lead to clonogenic inactivation and that chromosome aberrations are due to micrometre-scale rejoining of chromosome fragments derived from DNA cluster lesions (CLs). The CL yield and the threshold distance governing fragment rejoining were left as model parameters. The model, implemented as a Monte Carlo code called BIANCA (BIophysical ANalysis of Cell death and chromosome Aberrations), provided simulated survival curves that were compared with survival data on AG1522 and V79 cells exposed to different radiation types, including heavy ions. The agreement between simulation outcomes and experimental data suggests that lethal aberrations are likely to play an important role in cell killing not only for AG1522 cells exposed to X rays, as already reported by others, but also for other radiation types and other cells. Furthermore, the results are consistent with the hypothesis that the critical DNA lesions leading to cell death and chromosome aberrations are double-strand break clusters (possibly involving the ∼1000-10 000 bp scale) and that the effects of such clusters are modulated by micrometre-scale proximity effects during DNA damage processing.

Testing and linearity calibration of films of phenol compounds exposed to thermal neutron field for EPR dosimetry.

This paper reports the preliminary results obtained by Electron Paramagnetic Resonance (EPR) measurements on films of IRGANOX® 1076 phenols with and without low content (5% by weight) of gadolinium oxide (Gd2O3) exposed in the thermal column of the Triga Mark II reactor of LENA (Laboratorio Energia Nucleare Applicata) of Pavia (Italy). Thanks to their size, the phenolic films here presented are good devices for the dosimetry of beams with high dose gradient and which require accurate knowledge of the precise dose delivered. The dependence of EPR signal as function of neutron dose was investigated in the fluence range between 10(11) cm(-2) and 10(14) cm(-2). Linearity of EPR response was found and the signal was compared with that of commercial alanine films. Our analysis showed that gadolinium oxide (5% by weight) can enhance the thermal neutron sensitivity more than 18 times. Irradiated dosimetric films of phenolic compound exhibited EPR signal fading of about 4% after 10 days from irradiation.

Evaluation of the dose enhancement of combined ¹°B + ¹57Gd neutron capture therapy (NCT).

An innovative molecule, GdBLDL, for boron neutron capture therapy (BNCT) has been developed and its effectiveness as a BNCT carrier is currently under evaluation using in vivo experiments on small animal tumour models. The molecule contains both (10)B (the most commonly used NCT agent) and (157)Gd nuclei. (157)Gd is the second most studied element to perform NCT, mainly thanks to its high cross section for the capture of low-energy neutrons. The main drawback of (157)Gd neutron capture reaction is the very short range and low-energy secondary charged particles (Auger electrons), which requires (157)Gd to be very close to the cellular DNA to have an appreciable biological effect. Treatment doses were calculated by Monte Carlo simulations to ensure the optimised tumour irradiation and the sparing of the healthy organs of the irradiated animals. The enhancement of the absorbed dose due to the simultaneous presence of (10)B and (157)Gd in the experimental set-up was calculated and the advantage introduced by the presence of (157)Gd was discussed.

Comparative study of the radiobiological effects induced on adherent vs suspended cells by BNCT, neutrons and gamma rays treatments.

The present work is part of a preclinical in vitro study to assess the efficacy of BNCT applied to liver or lung coloncarcinoma metastases and to limb osteosarcoma. Adherent growing cell lines can be irradiated as adherent to the culture flasks or as cell suspensions, differences in radio-sensitivity of the two modalities of radiation exposure have been investigated. Dose related cell survival and cell cycle perturbation results evidenced that the radiosensitivity of adherent cells is higher than that of the suspended ones.

Toward a clinical application of ex situ boron neutron capture therapy for lung tumors at the RA-3 reactor in Argentina.

PURPOSE: Many types of lung tumors have a very poor prognosis due to their spread in the whole organ volume. The fact that boron neutron capture therapy (BNCT) would allow for selective targeting of all the nodules regardless of their position, prompted a preclinical feasibility study of ex situ BNCT at the thermal neutron facility of RA-3 reactor in the province of Buenos Aires, Argentina. (l)-4p-dihydroxy-borylphenylalanine fructose complex (BPA-F) biodistribution studies in an adult sheep model and computational dosimetry for a human explanted lung were performed to evaluate the feasibility and the therapeutic potential of ex situ BNCT. METHODS: Two kinds of boron biodistribution studies were carried out in the healthy sheep: a set of pharmacokinetic studies without lung excision, and a set that consisted of evaluation of boron concentration in the explanted and perfused lung. In order to assess the feasibility of the clinical application of ex situ BNCT at RA-3, a case of multiple lung metastases was analyzed. A detailed computational representation of the geometry of the lung was built based on a real collapsed human lung. Dosimetric calculations and dose limiting considerations were based on the experimental results from the adult sheep, and on the most suitable information published in the literature. In addition, a workable treatment plan was considered to assess the clinical application in a realistic scenario. RESULTS: Concentration-time profiles for the normal sheep showed that the boron kinetics in blood, lung, and skin would adequately represent the boron behavior and absolute uptake expected in human tissues. Results strongly suggest that the distribution of the boron compound is spatially homogeneous in the lung. A constant lung-to-blood ratio of 1.3 ± 0.1 was observed from 80 min after the end of BPA-F infusion. The fact that this ratio remains constant during time would allow the blood boron concentration to be used as a surrogate and indirect quantification of the estimated value in the explanted healthy lung. The proposed preclinical animal model allowed for the study of the explanted lung. As expected, the boron concentration values fell as a result of the application of the preservation protocol required to preserve the lung function. The distribution of the boron concentration retention factor was obtained for healthy lung, with a mean value of 0.46 ± 0.14 consistent with that reported for metastatic colon carcinoma model in rat perfused lung. Considering the human lung model and suitable tumor control probability for lung cancer, a promising average fraction of controlled lesions higher than 85% was obtained even for a low tumor-to-normal boron concentration ratio of 2. CONCLUSIONS: This work reports for the first time data supporting the validity of the ovine model as an adequate human surrogate in terms of boron kinetics and uptake in clinically relevant tissues. Collectively, the results and analysis presented would strongly suggest that ex situ whole lung BNCT irradiation is a feasible and highly promising technique that could greatly contribute to the treatment of metastatic lung disease in those patients without extrapulmonary spread, increasing not only the expected overall survival but also the resulting quality of life.

A pilot survey of cystic fibrosis clinical manifestations in CFTR mutation heterozygotes.

A cystic fibrosis (CF) heterozygote incidence higher than in the general population has been repeatedly reported in conditions which include clinical features found in CF, like pancreatitis, disseminated bronchiectasis, and allergic bronchopulmonary aspergillosis. Some cases may be explained by an unidentified compound heterozygosity; others could be manifesting heterozygotes. This study was aimed at detecting the incidence of CF-related clinical features in a population of carriers. A group of 261 obligate heterozygotes (mean age, 44 years) and a control group, composed of 201 individuals negative for a standard mutation panel (mean age, 36 years), were surveyed for possibly CF-related conditions (asthma, bronchiectasis, pneumothorax, allergic bronchopulmonary aspergillosis, sinusitis, nasal polyps, gallstones, liver cirrhosis, diabetes, pancreatitis, bone fractures, plus hypertension). A questionnaire was administered, and the accuracy of the statements was evaluated by phone interviews. There was no difference between heterozygotes and controls, with the exception of hypertension (carriers 28/261, controls 7/201, p = 0.004), and, in males, nasal polyps (carriers 7/126, controls 0/102, p value = 0.0178), and, again, hypertension (carriers 17/126, controls 5/102, p value = 0.0407). To avoid age bias, 126 heterozygotes matched to controls of the same gender and age were separately processed: these two groups showed no significant differences. CF-related clinical manifestations in obligate CFTR mutation heterozygotes are not more represented than in individuals with a low risk of being carriers.

How to study boron biodistribution in liver metastases from colorectal cancer.

The purpose of this study was to evaluate boron distribution for a safe and effective BNCT (Boron Neutron Capture Therapy) of liver metastases. Samples both from healthy and tumour liver parenchyma were analysed, after i.v. boron administration, by: alpha particles counting under neutron irradiation; morphological analysis by standard haematoxylin-eosin staining; neutron autoradiography. Our method was unaffected by the cytological heterogeneity inside tumour nodules; it demonstrated selective boron distribution in tumour tissue and predicted estimated mean therapeutic doses in tumour and safety doses in healthy tissue. The time interval for efficient BNCT was 2 to 4 hours after i.v. boron administration.

Simulations of neutron transport at low energy: a comparison between GEANT and MCNP.

The use of the simulation tool GEANT for neutron transport at energies below 20 MeV is discussed, in particular with regard to shielding and dose calculations. The reliability of the GEANT/MICAP package for neutron transport in a wide energy range has been verified by comparing the results of simulations performed with this package in a wide energy range with the prediction of MCNP-4B, a code commonly used for neutron transport at low energy. A reasonable agreement between the results of the two codes is found for the neutron flux through a slab of material (iron and ordinary concrete), as well as for the dose released in soft tissue by neutrons. These results justify the use of the GEANT/MICAP code for neutron transport in a wide range of applications, including health physics problems.

Microdosimetric measurements in the thermal neutron irradiation facility of LENA reactor.

A twin TEPC with electric-field guard tubes has been constructed to be used to characterize the BNCT field of the irradiation facility of LENA reactor. One of the two mini TEPC was doped with 50ppm of (10)B in order to simulate the BNC events occurring in BNCT. By properly processing the two microdosimetric spectra, the gamma, neutron and BNC spectral components can be derived with good precision (~6%). However, direct measurements of (10)B in some doped plastic samples, which were used for constructing the cathode walls, point out the scarce accuracy of the nominal (10)B concentration value. The influence of the Boral(®) door, which closes the irradiation channel, has been measured. The gamma dose increases significantly (+51%) when the Boral(®) door is closed. The crypt-cell-regeneration weighting function has been used to measure the quality, namely the RBEµ value, of the radiation field in different conditions. The measured RBEµ values are only partially consistent with the RBE values of other BNCT facilities.

Biodistribution of the boron carriers boronophenylalanine (BPA) and/or decahydrodecaborate (GB-10) for Boron Neutron Capture Therapy (BNCT) in an experimental model of lung metastases.

BNCT was proposed for the treatment of diffuse, non-resectable tumors in the lung. We performed boron biodistribution studies with 5 administration protocols employing the boron carriers BPA and/or GB-10 in an experimental model of disseminated lung metastases in rats. All 5 protocols were non-toxic and showed preferential tumor boron uptake versus lung. Absolute tumor boron concentration values were therapeutically useful (25-76ppm) for 3 protocols. Dosimetric calculations indicate that BNCT at RA-3 would be potentially therapeutic without exceeding radiotolerance in the lung.

Differential serotonin transport is linked to the rh5-HTTLPR in peripheral blood cells.

The human serotonin transporter (SERT) gene possesses a 43-base pair (bp) insertion-deletion promoter polymorphism, the h5-HTTLPR. Genotype at this locus correlates with variation in anxiety-related personality traits and risk for major depressive disorder in many studies. Yet, the complex effects of the h5-HTTLPR, in combination with closely associated single-nucleotide polymorphisms (SNPs), continue to be debated. Moreover, although SERT is of high clinical significance, transporter function in vivo remains difficult to assess. Rhesus express a promoter polymorphism related to the h5-HTTLPR. The rh5-HTTLPR has been linked to differences in stress-related behavior and cognitive flexibility, although allelic variations in serotonin uptake have not been investigated. We studied the serotonin system as it relates to the 5-HTTLPR in rhesus peripheral blood cells. Sequencing of the rh5-HTTLPR revealed a 23-bp insertion, which is somewhat longer than originally reported. Consistent with previous reports, no SNPs in the rh5-HTTLPR and surrounding genomic regions were detected in the individuals studied. Reductions in serotonin uptake rates, cell surface SERT binding, and 5-hydroxyindoleacetic acid/serotonin ratios, but not SERT mRNA levels, were associated with the rh5-HTTLPR short allele. Thus, serotonin uptake rates are differentiable with respect to the 5-HTTLPR in an easily accessible native peripheral tissue. In light of these findings, we foresee that primary blood cells, in combination with high sensitivity functional measurements enabled by chronoamperometry, will be important for investigating alterations in serotonin uptake associated with genetic variability and antidepressant responsiveness in humans.

From radiation-induced chromosome damage to cell death: modelling basic mechanisms and applications to boron neutron capture therapy.

Cell death is a crucial endpoint in radiation-induced biological damage: on one side, cell death is a reference endpoint to characterise the action of radiation in biological targets; on the other side, any cancer therapy aims to kill tumour cells. Starting from Lea's target theory, many models have been proposed to interpret radiation-induced cell killing; after briefly discussing some of these models, in this paper, a mechanistic approach based on an experimentally observed link between chromosome aberrations and cell death was presented. More specifically, a model and a Monte Carlo code originally developed for chromosome aberrations were extended to simulate radiation-induced cell death applying an experimentally observed one-to-one relationship between the average number of 'lethal aberrations' (dicentrics, rings and deletions) per cell and -ln S, S being the fraction of surviving cells. Although such observation was related to X rays, in the present work, the approach was also applied to protons and alpha particles. A good agreement between simulation outcomes and literature data provided a model validation for different radiation types. The same approach was then successfully applied to simulate the survival of cells enriched with boron and irradiated with thermal neutrons at the Triga Mark II reactor in Pavia, to mimic a typical treatment for boron neutron capture therapy.

Structure and stability of nickel/nickel oxide core-shell nanoparticles.

The results of a combined x-ray photoelectron spectroscopy (XPS) and high resolution transmission electron microscopy (HR-TEM) study of Ni nanoparticles (NP), before and after oxidation, are presented. An experimental set-up was realized for the preparation and study of pre-formed NP films, concentrating the attention on Ni NP in the diameter range between 4 and 8 nm. The XPS data were taken in situ from NPs after different stages of oxidation, including controlled dosing of O(2) gas in the experimental system and exposure to the atmosphere. The Ni 2p structure is a combination of spectra from metallic Ni in the NP core and from the oxide shell. The signal from the NP core was observed even for samples after exposure to air. From the comparison of HR-TEM experimental images with theoretical simulations, it was found that the Ni NP core has a regular multitwinned icosahedral structure, composed of single-crystal tetrahedra with (111) faces. The NiO phase is clearly observed forming islands on the NP surface.

Dose estimation in B16 tumour bearing mice for future irradiation in the thermal column of the TRIGA reactor after B/Gd/LDL adduct infusion.

To test the efficacy of a new (10)B-vector compound, the B/Gd/LDL adduct synthesised at Torino University, in vivo irradiations of murine tumours are in progress at the TRIGA Mark II reactor of the Pavia University. A localised B16 melanoma tumour is generated in C57BL/6 mice and subsequently infused with the adduct. During the irradiation, the mouse will be put in a shield to protect the whole body except the tumour in the back-neck area. To optimise the treatment set-up, MCNP simulations were performed. A very simplified mouse model was built using MCNP geometry capabilities, as well as the geometry of the shield made of 99% (10)B enriched boric acid. A hole in the shield is foreseen in correspondence of the back-neck region. Many configurations of the shield were tested in terms of neutron flux, dose distribution and mean induced activity in the tumour region and in the radiosensitive organs of the mouse. In the final set-up, up to five mice can be treated simultaneously in the reactor thermal column and the neutron fluence in the tumour region for 10 min of irradiation is of about 5×10(12) cm(-2).

A micro-PET/CT approach using O-(2-[18F]fluoroethyl)-L-tyrosine in an experimental animal model of F98 glioma for BNCT.

The present study focuses on a micro-PET/CT application to be used for experimental Boron Neutron Capture Therapy (BNCT), which integrates, in the same frame, micro-CT derived anatomy and PET radiotracer distribution. Preliminary results have demonstrated that (18)F-fluoroethyl-tyrosine (FET)/PET allows the identification of the extent of cerebral lesions in F98 tumor bearing rat. Neutron autoradiography and α-spectrometry on axial tissues slices confirmed the tumor localization and extraction, after the administration of fructose-boronophenylalanine (BPA). Therefore, FET-PET approach can be used to assess the transport, the net influx, and the accumulation of FET, as an aromatic amino acid analog of BPA, in experimental animal model. Coregistered micro-CT images allowed the accurate morphological localization of the radiotracer distribution and its potential use for experimental BNCT.

Cell death following BNCT: a theoretical approach based on Monte Carlo simulations.

In parallel to boron measurements and animal studies, investigations on radiation-induced cell death are also in progress in Pavia, with the aim of better characterisation of the effects of a BNCT treatment down to the cellular level. Such studies are being carried out not only experimentally but also theoretically, based on a mechanistic model and a Monte Carlo code. Such model assumes that: (1) only clustered DNA strand breaks can lead to chromosome aberrations; (2) only chromosome fragments within a certain threshold distance can undergo misrejoining; (3) the so-called "lethal aberrations" (dicentrics, rings and large deletions) lead to cell death. After applying the model to normal cells exposed to monochromatic fields of different radiation types, the irradiation section of the code was purposely extended to mimic the cell exposure to a mixed radiation field produced by the (10)B(n,α) (7)Li reaction, which gives rise to alpha particles and Li ions of short range and high biological effectiveness, and by the (14)N(n,p)(14)C reaction, which produces 0.58 MeV protons. Very good agreement between model predictions and literature data was found for human and animal cells exposed to X- or gamma-rays, protons and alpha particles, thus allowing to validate the model for cell death induced by monochromatic radiation fields. The model predictions showed good agreement also with experimental data obtained by our group exposing DHD cells to thermal neutrons in the TRIGA Mark II reactor of the University of Pavia; this allowed to validate the model also for a BNCT exposure scenario, providing a useful predictive tool to bridge the gap between irradiation and cell death.