Radiation Track Structure: How the Spatial Distribution of Energy Deposition Drives Biological Response.

Ionising radiation is incredibly effective at causing biological effects. This is due to the unique way energy is deposited along highly structured tracks of ionisation and excitation events, which results in correlation with sites of DNA damage from the nanometre to the micrometre scale. Correlation of these events along the track on the nanometre scale results in clustered damage, which not only results in the formation of DNA double-strand breaks (DSB), but also more difficult to repair complex DSB, which include additional damage within a few base pairs. The track structure varies significantly with radiation quality and the increase in relative biological effectiveness observed with increasing linear energy transfer in part corresponds to an increase in the probability and complexity of clustered DNA damage produced. Likewise, correlation over larger scales, associated with packing of DNA and associated chromosomes within the cell nucleus, can also have a major impact on the biological response. The proximity of the correlated damage along the track increases the probability of miss-repair through pairwise interactions resulting in an increase in probability and complexity of DNA fragments/deletions, mutations and chromosomal rearrangements. Understanding the mechanisms underlying the biological effectiveness of ionising radiation can provide an important insight into ways of increasing the efficacy of radiotherapy, as well as the risks associated with exposure. This requires a multi-scale approach for modelling, not only considering the physics of the track structure from the millimetre scale down to the nanometre scale, but also the structural packing of the DNA within the nucleus, the resulting chemistry in the context of the highly reactive environment of the nucleus, together with the subsequent biological response.

The Development of Technology for Effective Respiratory-Gated Irradiation Using an Image-Guided Small Animal Irradiator.

The development of image-guided small animal irradiators represents a significant improvement over standard irradiators by enabling preclinical studies to mimic radiotherapy in humans. The ability to deliver tightly collimated targeted beams, in conjunction with gantry or animal couch rotation, has the potential to maximize tumor dose while sparing normal tissues. However, the current commercial platforms do not incorporate respiratory gating, which is required for accurate and precise targeting in organs subject to respiration related motions that may be up to the order of 5 mm in mice. Therefore, a new treatment head assembly for the Xstrahl Small Animal Radiation Research Platform (SARRP) has been designed. This includes a fast X-ray shutter subsystem, a motorized beam hardening filter assembly, an integrated transmission ionization chamber to monitor beam delivery, a kinematically positioned removable beam collimator and a targeting laser exiting the center of the beam collimator. The X-ray shutter not only minimizes timing errors but also allows beam gating during imaging and treatment, with irradiation only taking place during the breathing cycle when tissue movement is minimal. The breathing related movement is monitored by measuring, using a synchronous detector/lock-in amplifier that processes diffuse reflectance light from a modulated light source. After thresholding of the resulting signal, delays are added around the inhalation/exhalation phases, enabling the "no movement" period to be isolated and to open the X-ray shutter. Irradiation can either be performed for a predetermined time of X-ray exposure, or through integration of a current from the transmission monitor ionization chamber (corrected locally for air density variations). The ability to successfully deliver respiratory-gated X-ray irradiations has been demonstrated by comparing movies obtained using planar X-ray imaging with and without respiratory gating, in addition to comparing dose profiles observed from a collimated beam on EBT3 radiochromic film mounted on the animal's chest. Altogether, the development of respiratory-gated irradiation facilitates improved dose delivery during animal movement and constitutes an important new tool for preclinical radiation studies. This approach is particularly well suited for irradiation of orthotopic tumors or other targets within the chest and abdomen where breathing related movement is significant.

Comments on potential health effects of MRI-induced DNA lesions: quality is more important to consider than quantity.

Magnetic resonance imaging (MRI) is increasingly being used in cardiology to detect heart disease and guide therapy. It is mooted to be a safer alternative to imaging techniques, such as computed tomography (CT) or coronary angiographic imaging. However, there has recently been an increased interest in the potential long-term health risks of MRI, especially in the light of the controversy resulting from a small number of research studies reporting an increase in DNA damage following exposure, with calls to limit its use and avoid unnecessary examination, according to the precautionary principle. Overall the published data are somewhat limited and inconsistent; the ability of MRI to produce DNA lesions has yet to be robustly demonstrated and future experiments should be carefully designed to optimize sensitivity and benchmarked to validate and assess reproducibility. The majority of the current studies have focussed on the initial induction of DNA damage, and this has led to comparisons between the reported induction of γH2AX and implied double-strand break (DSB) yields produced following MRI with induction by imaging techniques using ionizing radiation. However, γH2AX is not only a marker of classical double-ended DSB, but also a marker of stalled replication forks and in certain circumstances stalled DNA transcription. Additionally, ionizing radiation is efficient at producing complex DNA damage, unique to ionizing radiation, with an associated reduction in repairability. Even if the fields associated with MRI are capable of producing DNA damage, the lesions produced will in general be simple, similar to those produced by endogenous processes. It is therefore inappropriate to try and infer cancer risk by simply comparing the yields of γH2AX foci or DNA lesions potentially produced by MRI to those produced by a given exposure of ionizing radiation, which will generally be more biologically effective and have a greater probability of leading to long-term health effects. As a result, it is important to concentrate on more relevant downstream end points (e.g. chromosome aberration production), along with potential mechanisms by which MRI may lead to DNA lesions. This could potentially involve a perturbation in homeostasis of oxidative stress, modifying the background rate of endogenous DNA damage induction. In summary, what the field needs at the moment is more research and less fear mongering.

Fishing for radiation quality: chromosome aberrations and the role of radiation track structure.

The yield of chromosome aberrations is not only dependent on dose but also on radiation quality, with high linear energy transfer (LET) typically having a greater biological effectiveness per unit dose than those of low-LET radiation. Differences in radiation track structure and cell morphology can also lead to quantitative differences in the spectra of the resulting chromosomal rearrangements, especially at low doses associated with typical human exposures. The development of combinatorial fluorescent labelling techniques (such as mFISH and mBAND) has helped to reveal the complexity of rearrangements, showing increasing complexity of observed rearrangements with increasing LET but has a resolution limited to ∼10 MBp. High-LET particles have not only been shown to produce clustered sites of DNA damage but also produce multiple correlated breaks along its path resulting in DNA fragments smaller than the resolution of these techniques. Additionally, studies have shown that the vast majority of radiation-induced HPRT mutations were also not detectable using fluorescent in situ hybridisation (FISH) techniques, with correlation of breaks along the track being reflected in the complexity of mutations, with intra- and inter-chromosomal insertions, and inversions occurring at the sites of some of the deletions. Therefore, the analysis of visible chromosomal rearrangements observed using current FISH techniques is likely to represent just the tip of the iceberg, considerably underestimating the extent and complexity of radiation induced rearrangements.

Stimulation of intercellular induction of apoptosis in transformed cells at very low doses of ionising radiation: spatial and temporal features.

The ultimate response of a cell or tissue to radiation is dependent in part on intercellular signalling. This becomes increasingly important at low doses, or at low dose rates, associated with typical human exposures. In order to help characterise the underlying mechanism of intercellular signalling, and how they are perturbed following exposure to ionising radiation, a previously well-defined model system of intercellular induction of apoptosis (IIA) (Portess et al. 2007, Cancer Res. 67, 1246-1253) was adopted. The aim of the present work is to evaluate the signalling mechanisms underpinning this process through exploring the variables that can affect the IIA, i.e. dose, time and space.

Non-targeted effects of radiation exposure: recent advances and implications.

The target theory of radiation-induced effects has been challenged by numerous studies, which indicate that in addition to biological effects resulting from direct DNA damage within the cell, a variety of non-DNA targeted effects (NTE) may make important contributions to the overall outcome. Ionising radiation induces complex, global cellular responses, such as genomic instability (GI) in both irradiated and never-irradiated 'bystander' cells that receive molecular signals produced by irradiated cells. GI is a well-known feature of many cancers, increasing the probability of cells to acquire the 'hallmarks of cancer' during the development of tumours. Although epidemiological data include contributions of both direct and NTE, they lack (i) statistical power at low dose where differences in dose response for NTE and direct effects are likely to be more important and (ii) heterogeneity of non-targeted responses due to genetic variability between individuals. In this article, NTE focussing on GI and bystander effects were critically examined, the specific principles of NTE were discussed and the potential influence on human health risk assessment from low-dose radiation was considered.

Validation of a prototype DiodeAir for small field dosimetry.

Standard commercial diode detectors over-respond within small radiation fields, an effect largely attributable to the relatively high mass-density of silicon. However, Monte Carlo studies can be used to optimise dosimeter designs and have demonstrated that 'mass-density compensation'-for example, introducing a low-density air-gap upstream of a diode's high-density silicon volume-can substantially improve instrument response. In this work we used egs_chamber Monte Carlo simulations to predict the ideal air-gap thickness for a PTW 60017 unshielded diode detector. We then developed a prototype instrument incorporating that air-gap and, for a 6 MV linac, tested it experimentally against EBT3 film. We also tested a further three prototypes with different air-gap thicknesses. Our results demonstrate that for a 10 × 10 cm(2) reference field the DiodeAir, a PTW 60017 diode with a built-in air-gap of 1 mm, has on-axis correction factors near unity. Laterally the DiodeAir performs very well off-axis and reports FWHM and penumbra values consistent with those measured using EBT3. For PDD measurement, the performance of the DiodeAir matches that of the original PTW 60017. The experimental focus of this work was 6 MV but we also simulated the on-axis response of the DiodeAir within 15 MV beams and found that our modification proved robust to this substantial increase in beam energy. However, the original diode 60017 does exhibit low energy scatter dependencies and may over-respond to high linac dose-rates such that applying the mass-density compensation method to an alternative instrument (particularly a diamond detector) could ultimately take us even closer to the small-field ideal.

Under-response correction for EBT3 films in the presence of proton spread out Bragg peaks.

We present a study of the under-response of the new Gafchromic EBT3 films and a procedure to accurately perform 2D and 3D proton dosimetry measurements for both pristine and spread out Bragg peaks (SOBP) of any energy. These new films differ from the previous EBT2 generation by a slightly different active layer composition, which we show has not effected appreciably their response. The procedure and the beam quality correction factor curve have been benchmarked using 29 MeV modulated proton beams. In order to show the correction to apply when EBT3 films are used as treatment verification tools in anthropomorphic phantoms, two simulation studies involving clinical energies are presented: a SOBP for eye treatments and a SOBP to treat 20 cm deep and 5 cm thick tumours. We find maximum under-responses of 37%, 30% and 7.7% for the modulated 29 MeV beam, eye and deep tumour treatment, respectively, which were attained close to the end of the peak tails, due to a higher proportion of very low energy protons. The maximum deviations between corrected and uncorrected doses were for the three cases, respectively, 20.7%, 8.3% and 2.1% of the average dose across flat region of the SOBP. These values were obtained close to the distal edge of the SOBPs, where the proportion of low energy protons was not as high as on the tail, but there still was a number of protons high enough to deposit a reasonable amount of dose in the films.

Mass-density compensation can improve the performance of a range of different detectors under non-equilibrium conditions.

Dosimeters often consist of several components whose mass densities differ substantially from water. These components cause small-field correction factors to vary significantly as lateral electronic equilibrium breaks down. Even amongst instruments designed for small-field dosimetry, inter-detector variation in the correction factors associated with very small (∼0.5 cm) fields can amount to tens of per cent. For a given dosimeter, small-field correction factors vary not only with field size but also with detector azimuthal angle and position within the field. Furthermore the accurate determination of these factors typically requires time-intensive Monte Carlo simulations. Thus, if achievable, 'correction factor free' small-field dosimetry would be highly desirable. This study demonstrates that a new generation of mass-density compensated detectors could take us towards this goal. Using a 6 MV beam model, it shows that 'mass-density compensation' can be utilized to improve the performance of a range of different detectors under small-field conditions. Non-sensitive material of appropriate mass-density is incorporated into detector designs in order to make the instruments behave as if consisting only of water. The dosimeter perturbative effects are then reduced to those associated with volume averaging. An even better solution-which modifies detectors to obtain profiles that look like those measured by a point-like water structure-is also considered. Provided that adequate sensitivity can be achieved for a small measurement volume, this study shows that it may be possible to use mass-density compensation (and Monte Carlo-driven design) to produce a solid-state dosimeter/ionization chamber with a near-perfect non-equilibrium response.

Detector density and small field dosimetry: integral versus point dose measurement schemes.

PURPOSE: The Alfonso et al. [Med. Phys. 35, 5179-5186 (2008)] formalism for small field dosimetry proposes a set of correction factors (kQclin,Qmsrfclin,fmsr) which account for differences between the detector response in nonstandard (clinical) and machine-specific-reference fields. In this study, the Monte Carlo method was used to investigate the viability of such small field correction factors for four different detectors irradiated under a variety of conditions. Because kQclin,Qmsrfclin,fmsr values for single detector position measurements are influenced by several factors, a new theoretical formalism for integrated-detector-position [dose area product (DAP)] measurements is also presented and was tested using Monte Carlo simulations. METHODS: A BEAMnrc linac model was built and validated for a Varian Clinac iX accelerator. Using the egs++ geometry package, detailed virtual models were built for four different detectors: a PTW 60012 unshielded diode, a PTW 60003 Diamond detector, a PTW 31006 PinPoint (ionization chamber), and a PTW 31018 MicroLion (liquid-filled ionization chamber). The egs_chamber code was used to investigate the variation of kQclin,Qmsrfclin,fmsr with detector type, detector construction, field size, off-axis position, and the azimuthal angle between the detector and beam axis. Simulations were also used to consider the DAP obtained by each detector: virtual detectors and water voxels were scanned through high resolution grids of positions extending far beyond the boundaries of the fields under consideration. RESULTS: For each detector, the correction factor (kQclin,Qmsrfclin,fmsr) was shown to depend strongly on detector off-axis position and detector azimuthal angle in addition to field size. In line with previous studies, substantial interdetector variation was also observed. However, it was demonstrated that by considering DAPs rather than single-detector-position dose measurements the high level of interdetector variation could be eliminated. Under small field conditions, mass density was found to be the principal determinant of water equivalence. Additionally, the mass densities of components outside the sensitive volumes were found to influence the detector response. CONCLUSIONS: kQclin,Qmsrfclin,fmsr values for existing detector designs depend on a host of variables and their calculation typically relies on the use of time-intensive Monte Carlo methods. Future moves toward density-compensated detector designs or DAP based protocols may simplify the methodology of small field dosimetry.

Chromosome aberration induction is dependent on the spatial distribution of energy deposition through a cell nucleus.

The importance of the spatial distribution of energy deposition through the nucleus in determining the resultant chromosome rearrangements was investigated using fluorescent in situ hybridisation technique following either uniform or partial irradiation of HF19 human fibroblast cells with low-LET 1.5 keV ultrasoft X-rays. Irradiations were performed with and without a copper irradiation mask with a Poisson distribution of micron-sized holes immediately below the irradiation dish and the results are compared with previous results obtained following exposure to a Poisson distribution of alpha particles. For the same radiation quality, the spatial distribution of energy deposition within the nucleus was found to be important in determining the ultimate biological response, with an increased ratio of complex-to-simple aberrations observed for partial compared to uniform irradiation. Comparisons between low-LET ultrasoft X-rays and high-LET alpha particles indicate that the sub-micron clustering of damage along the alpha particle track is more important than just the total number of double-strand breaks produced.

Reduced EDHF responses and connexin activity in mesenteric arteries from the insulin-resistant obese Zucker rat.

AIMS/HYPOTHESIS: The objective of this study was to examine the effect of insulin resistance on endothelium-derived hyperpolarising factor (EDHF) and small mesenteric artery endothelial function using 25-week-old insulin-resistant obese Zucker rats (OZRs) and lean littermate control rats (LZRs). The involvement of gap junctions and their connexin subunits in the EDHF relaxation response was also assessed. METHODS: Mesenteric arteries were evaluated using the following assays: (1) endothelial function by pressure myography, with internal diameter recorded using video microscopy; (2) connexin protein levels by western blotting; and (3) Cx mRNA expression by real-time PCR. RESULTS: Relaxations in response to acetylcholine were significantly smaller in mesenteric arteries from the OZRs than the LZRs, whereas there was no difference in relaxations in response to levcromakalim. Responses to acetylcholine were not altered by nitric oxide inhibitors, but were abolished by charybdotoxin in combination with apamin, which blocked the EDHF component of the response. 40Gap27 significantly attenuated the response to acetylcholine in the LZRs, but had no effect in the OZRs. Connexin 40 protein and Cx40 mRNA levels in mesenteric vascular homogenates were significantly smaller in the OZRs than in the LZRs, with no difference in connexin 43 or Cx43 mRNA levels. CONCLUSIONS/INTERPRETATION: These findings demonstrate that endothelial dysfunction in mesenteric arteries from the insulin-resistant OZRs can be attributed to a defect in EDHF. The results also suggest that the defective EDHF is at least partly related to an impairment of connexin 40-associated gap junctions, through a decrease in connexin 40 protein and Cx40 mRNA expression in the OZRs.

Endothelium-independent constriction of isolated, pressurized arterioles by Nomega-nitro-L-arginine methyl ester (L-NAME).

BACKGROUND AND PURPOSE: Nitric oxide synthase (NOS) inhibitors cause vasoconstriction in pressurized arterioles with myogenic tone. This suggests either tonic production of NO modulates myogenic tone or a direct, NOS-independent effect of the NOS inhibitors. The nature of the contractile effect of the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM) on pressurised arterioles was investigated. EXPERIMENTAL APPROACH: Segments of rat cremaster muscle first-order arteriole were cannulated on glass micropipettes and maintained at an intraluminal pressure of 50, 70 or 120 mmHg. KEY RESULTS: L-NAME and the related compound L-NA (100 microM) constricted pressurized vessels with myogenic tone. Removal of the endothelium did not cause constriction or alter myogenic tone, however the constrictor effect of L-NAME persisted. The constrictor effect of L-NAME was abolished by L-arginine (1 mM). Other NO and cGMP pathway inhibitors, including the nNOS inhibitor 7-nitroindazole (100 muM), the NO scavenger carboxy-PTIO (100 microM), the guanylate cyclase inhibitor ODQ (10 microM) and the cGMP inhibitor Rp-8CPT-cGMPS (10 microM) did not cause constriction of the arterioles. L-NAME caused a small (3-4 mV) but not statistically significant depolarization of the arteriolar smooth muscle at both pressures. The constrictor effect was not prevented by the K(+)-channel antagonist tetraethyl ammonium (TEA, 1 mM) or the K(ATP) channel antagonist glibenclamide (1 microM). CONCLUSIONS AND IMPLICATIONS: These observations demonstrate that L-NAME causes an endothelium- and NOS-independent contraction of vascular smooth muscle in isolated skeletal muscle arterioles. It is suggested that the underlying mechanism relates to an arginine binding interaction.

Genomic instability and the role of radiation quality.

Genomic instability (GI) is a hallmark of tumorigenic progression and is observed as delayed genetic damage in the progeny of irradiated and unirradiated bystander cells. The expression of GI can be influenced by genotype, cell type and radiation quality. While several studies have demonstrated the induction of GI by high and low-linear energy transfer (LET) radiation, our work on human and mouse primary cell systems has shown LET-dependent differences in the induction and expression of GI. These differences might be attributed to differences in radiation track structure, dose rate, contribution of bystander cells and radiation dose. This paper reviews the role of radiation quality in the induction of GI and describe the possible mechanisms underlining the observed differences between radiation types on its induction. The experimental results presented suggest that dose might be the most significant factor in determining induction of GI after low-LET radiation.

Experimental techniques for studying bystander effects in vitro by high and low-LET ionising radiation.

Ionising radiation can induce responses within non-exposed neighbouring (bystander) cells which potentially have important implications on the estimates of risk from low dose or low dose rate exposures of ionising radiations. A range of strategies have been developed for investigating bystander effects in vitro for both high-LET alpha particles or low-LET ultrasoft X rays using either partial shielding (grids, half-shields and slits) or by using a co-culture system where two physically separated populations of cells can be cultured together, allowing one population of cells to be irradiated while the second population remains unirradiated. The techniques described provide a useful tool to study bystander effects and complement microbeam studies. Studies using these systems show significant increases in the unirradiated bystander cells for various end points including the induction of chromosomal instability in haemopoetic stem cells and transformation in CGL1 cells.

Keeping up with the neighbours--measuring the bystander response.

Ionising radiation can induce responses within non-exposed neighbouring (bystander) cells, which potentially have important implications on the estimates of risk at environmentally relevant doses. Using human skin fibroblasts (AG1522), a range of methods were used to investigate the nature of the signal(s) arising from the exposed cells. The signal(s) can be transmitted by direct cell-cell communication (investigated by using partial dish irradiations) or by medium-borne factors (a co-culture system where two monolayers share the same medium but only one monolayer is exposed to ionising radiation). CDKN1A was found to be up-regulated in both directly exposed and non-exposed cells. The data suggest that direct cell-cell communication dominates for these confluent cells, with medium-borne factors also contributing.

Human ganglion cells express the alpha-2 adrenergic receptor: relevance to neuroprotection.

BACKGROUND/AIM: Alpha-2alpha adrenergic receptor (alpha(2)-AR) agonists are thought to be neuroprotective, preventing retinal ganglion cell death independent of pressure reduction. Previous studies have identified alpha(2)-ARs in rat retina. The authors aimed to demonstrate the presence and localisation of alpha(2)-ARs in human and rat retina and on the rat retinal ganglion cell line, RGC-5. METHODS: Seven postmortem human and three postmortem rat eyes were paraformaldehyde fixed and frozen. RGC-5 cells were also paraformaldehyde fixed. The expression of alpha(2A)-ARs was determined by antibody immunofluorescence. RESULTS: alpha(2A)-AR expression was identified in the human retina, on ganglion cells, and cells in the inner and outer nuclear layers (INL, ONL). Differential alpha(2A)-AR staining patterns in the INL and ONL suggest a further restriction to as yet unidentified neuronal subclasses. The RGC-5 cell line also expressed alpha(2A)-ARs in undifferentiated cells and an increased expression upon fully differentiated cells. CONCLUSION: alpha(2)-AR agonists in addition to their pressure lowering effects in the eye, may act directly upon retinal neurons, including retinal ganglion cells. The presence of alpha(2)-ARs on the RGC-5 cell line allows future investigation of these possible direct effects using in vitro glaucoma model systems.

Bound PCNA in nuclei of primary rat tracheal epithelial cells after exposure to very low doses of plutonium-238 alpha particles.

Bystander effects from ionizing radiation have been detailed for a number of cell systems and a number of end points. We wished to use a cell culture/ex vivo rat model of respiratory tissue to determine whether a bystander effect detected in culture could also be shown in a tissue. Examination by immunofluorescence techniques of tracheal cell cultures after exposure to very low doses of alpha particles revealed a large proportion of cells with proliferating cell nuclear antigen (PCNA) bound in their nuclei. PCNA was selected as an end point because it is involved in both DNA repair and the changes in cell cycle that are typical of many reported bystander effects. Maximum response can be detected in up to 28% of the cells in sub-confluent cultures with a dose of only 2 mGy. At this dose less than 2% of the cell nuclei have experienced a particle traversal and less than 6% of the cells have experienced an alpha-particle traversal through either their nucleus or some part of their cytoplasm. The hypothesis that this bystander response in nontargeted cells is mediated through secreted factor(s) is presented, and supporting evidence was found using partial irradiation and co-culture experiments. Examination of the effect with excised pieces of trachea demonstrated a response similar to that seen in culture.

The variation in biological effectiveness of X-rays and gamma rays with energy.

The ICRP has attributed the same relative risk for all low-LET (linear energy transfer) radiations, including X and gamma radiations of all energies. However, very low energy X-rays are expected to be more biologically effective, per unit absorbed dose, than high energy X-rays or gamma rays due to the production of lower energy secondary electrons, with a correspondingly higher LET. This increase in relative biological effectiveness (RBE) is also seen experimentally for a range of biological end-points, however, a wide range of RBE values have been reported. The assessment of risks is particularly important due to the use of low energy X-rays for mammography screening. A review of the published data on the variation in biological effectiveness with energy is presented here.