Proton Minibeam Radiation Therapy Reduces Side Effects in an In Vivo Mouse Ear Model.

PURPOSE: Proton minibeam radiation therapy is a novel approach to minimize normal tissue damage in the entrance channel by spatial fractionation while keeping tumor control through a homogeneous tumor dose using beam widening with an increasing track length. In the present study, the dose distributions for homogeneous broad beam and minibeam irradiation sessions were simulated. Also, in an animal study, acute normal tissue side effects of proton minibeam irradiation were compared with homogeneous irradiation in a tumor-free mouse ear model to account for the complex effects on the immune system and vasculature in an in vivo normal tissue model. METHODS AND MATERIALS: At the ion microprobe SNAKE, 20-MeV protons were administered to the central part (7.2 × 7.2 mm(2)) of the ear of BALB/c mice, using either a homogeneous field with a dose of 60 Gy or 16 minibeams with a nominal 6000 Gy (4 × 4 minibeams, size 0.18 × 0.18 mm(2), with a distance of 1.8 mm). The same average dose was used over the irradiated area. RESULTS: No ear swelling or other skin reactions were observed at any point after minibeam irradiation. In contrast, significant ear swelling (up to fourfold), erythema, and desquamation developed in homogeneously irradiated ears 3 to 4 weeks after irradiation. Hair loss and the disappearance of sebaceous glands were only detected in the homogeneously irradiated fields. CONCLUSIONS: These results show that proton minibeam radiation therapy results in reduced adverse effects compared with conventional homogeneous broad-beam irradiation and, therefore, might have the potential to decrease the incidence of side effects resulting from clinical proton and/or heavy ion therapy.

The influence of the channel size on the reduction of side effects in microchannel proton therapy.

The potential of proton microchannel radiotherapy to reduce radiation effects in the healthy tissue but to keep tumor control the same as in conventional proton therapy is further elucidated. The microchannels spread on their way to the tumor tissue resulting in different fractions of the healthy tissue covered with doses larger than the tumor dose, while the tumor gets homogeneously irradiated. The aim of this study was to evaluate the effect of increasing channel width on potential side effects in the normal tissue. A rectangular 180 × 180 µm(2) and two Gaussian-type dose distributions of σ = 260 µm and σ = 520 µm with an interchannel distance of 1.8 mm have been applied by 20-MeV protons to a 3D human skin model in order to simulate the widened channels and to compare the irradiation effects at different endpoints to those of a homogeneous proton irradiation. The number of protons applied was kept constant at all irradiation modes resulting in the same average dose of 2 Gy. All kinds of proton microchannel irradiation lead to higher cell viability and produce significantly less genetic damage than homogeneous proton irradiation, but the reduction is lower for the wider channel sizes. Our findings point toward the application of microchannel irradiation for clinical proton or heavy ion therapy to further reduce damage of normal tissues while maintaining tumor control via a homogeneous dose distribution inside the tumor.

Reduced side effects by proton microchannel radiotherapy: study in a human skin model.

The application of a microchannel proton irradiation was compared to homogeneous irradiation in a three-dimensional human skin model. The goal is to minimize the risk of normal tissue damage by microchannel irradiation, while preserving local tumor control through a homogeneous irradiation of the tumor that is achieved because of beam widening with increasing track length. 20 MeV protons were administered to the skin models in 10- or 50-µm-wide irradiation channels on a quadratic raster with distances of 500 µm between each channel (center to center) applying an average dose of 2 Gy. For comparison, other samples were irradiated homogeneously at the same average dose. Normal tissue viability was significantly enhanced after microchannel proton irradiation compared to homogeneous irradiation. Levels of inflammatory parameters, such as Interleukin-6, TGF-Beta, and Pro-MMP1, were significantly lower in the supernatant of the human skin tissue after microchannel irradiation than after homogeneous irradiation. The genetic damage as determined by the measurement of micronuclei in keratinocytes also differed significantly. This difference was quantified via dose modification factors (DMF) describing the effect of each irradiation mode relative to homogeneous X-ray irradiation, so that the DMF of 1.21 ± 0.20 after homogeneous proton irradiation was reduced to 0.23 ± 0.11 and 0.40 ± 0.12 after microchannel irradiation using 10- and 50-µm-wide channels, respectively. Our data indicate that proton microchannel irradiation maintains cell viability while significantly reducing inflammatory responses and genetic damage compared to homogeneous irradiation, and thus might improve protection of normal tissue after irradiation.

Cigarette smoke-induced transgenerational alterations in genome stability in cord blood of human F1 offspring.

The relevance of preconceptional and prenatal toxicant exposures for genomic stability in offspring is difficult to analyze in human populations, because gestational exposures usually cannot be separated from preconceptional exposures. To analyze the roles of exposures during gestation and conception on genomic stability in the offspring, stability was assessed via the Comet assay and highly sensitive, semiautomated confocal laser scans of γH2AX foci in cord, maternal, and paternal blood as well as spermatozoa from 39 families in Crete, Greece, and the United Kingdom. With use of multivariate linear regression analysis with backward selection, preconceptional paternal smoking (% tail DNA: P>0.032; γH2AX foci: P>0.018) and gestational maternal (% tail DNA: P>0.033) smoking were found to statistically significantly predict DNA damage in the cord blood of F1 offspring. Maternal passive smoke exposure was not identified as a predictor of DNA damage in cord blood, indicating that the effect of paternal smoking may be transmitted via the spermatozoal genome. Taken together, these studies reveal a role for cigarette smoke in the induction of DNA alterations in human F1 offspring via exposures of the fetus in utero or the paternal germline. Moreover, the identification of transgenerational DNA alterations in the unexposed F1 offspring of smoking-exposed fathers supports the claim that cigarette smoke is a human germ cell mutagen.

Differences in Phosphorylated Histone H2AX Foci Formation and Removal of Cells Exposed to Low and High Linear Energy Transfer Radiation.

The use of particle ion beams in cancer radiotherapy has a long history. Today, beams of protons or heavy ions, predominantly carbon ions, can be accelerated to precisely calculated energies which can be accurately targeted to tumors. This particle therapy works by damaging the DNA of tissue cells, ultimately causing their death. Among the different types of DNA lesions, the formation of DNA double strand breaks is considered to be the most relevant of deleterious damages of ionizing radiation in cells. It is well-known that the extremely large localized energy deposition can lead to complex types of DNA double strand breaks. These effects can lead to cell death, mutations, genomic instability, or carcinogenesis. Complex double strand breaks can increase the probability of mis-rejoining by NHEJ. As a consequence differences in the repair kinetics following high and low LET irradiation qualities are attributed mainly to quantitative differences in their contributions of the fast and slow repair component. In general, there is a higher contribution of the slow component of DNA double strand repair after exposure to high LET radiation, which is thought to reflect the increased amount of complex DNA double strand breaks. These can be accurately measured by the γ-H2AX assay, because the number of phosphorylated H2AX foci correlates well with the number of double strand breaks induced by low or / and high LET radiation.

Scanning irradiation device for mice in vivo with pulsed and continuous proton beams.

A technical set-up for irradiation of subcutaneous tumours in mice with nanosecond-pulsed proton beams or continuous proton beams is described and was successfully used in a first experiment to explore future potential of laser-driven particle beams, which are pulsed due to the acceleration process, for radiation therapy. The chosen concept uses a microbeam approach. By focusing the beam to approximately 100 × 100 µm(2), the necessary fluence of 10(9) protons per cm(2) to deliver a dose of 20 Gy with one-nanosecond shot in the Bragg peak of 23 MeV protons is achieved. Electrical and mechanical beam scanning combines rapid dose delivery with large scan ranges. Aluminium sheets one millimetre in front of the target are used as beam energy degrader, necessary for adjusting the depth-dose profile. The required procedures for treatment planning and dose verification are presented. In a first experiment, 24 tumours in mice were successfully irradiated with 23 MeV protons and a single dose of 20 Gy in pulsed or continuous mode with dose differences between both modes of 10%. So far, no significant difference in tumour growth delay was observed.

Relative biological effectiveness of pulsed and continuous 20 MeV protons for micronucleus induction in 3D human reconstructed skin tissue.

BACKGROUND AND PURPOSE: Laser accelerated radiotherapy is a prospect for cancer treatment with proton and/or carbon ion beams that is currently under fast development. In principal, ultra fast, high-energy laser pulses will lead to a "pulsed" delivery of the induced ion beam with pulse durations of 1ns and below, whereas conventional proton beams deriving from a cyclotron or synchrotron apply the dose within 100 ms ("continuous"). MATERIALS AND METHODS: A simulation of both irradiation modes could be established at the Munich tandem accelerator with a 20MeV proton beam, and a wide-field fast scanning system was implemented that allowed for application of up to 5 Gy per tissue voxel in a single pulse. The relative biological effectiveness (RBE) of pulsed and continuous modes of irradiation with 20 MeV protons relative to the reference radiation 70 kV X-rays was examined in a human tissue model (3D human reconstructed skin, EpiDermFT) which preserves the three-dimensional geometric arrangement and communication of cells present in tissues in vivo. Using the induction of micronuclei (MN) in keratinocytes as the biological endpoint, the RBE was calculated as the ratio between the dose of 70 kV X-rays and 3 Gy of 20 MeV protons (pulsed or continuous) which produced equal response. RESULTS: For pulsed and continuous 20 MV proton exposures of the human skin model, RBE values of 1.08+/-0.20 and 1.22+/-0.15 versus 70 kV X-rays were obtained in a first experiment and 1.00+/-0.14 and 1.13+/-0.14 in a second experiment during distinct beam access times, respectively. The approximately 10% difference in RBE between the respective irradiation modes in both experiments was associated with large uncertainties which were not statistically significant (p approximately 0.5). CONCLUSION: These findings represent an important step on the way towards application of laser-accelerated protons for clinical radiotherapy. Further clinically relevant endpoints in normal and tumor tissue have to be evaluated.

Application of information-theoretic measures to quantitative analysis of immunofluorescent microscope imaging.

The goal of this contribution is to apply model-based information-theoretic measures to the quantification of relative differences between immunofluorescent signals. Several models for approximating the empirical fluorescence intensity distributions are considered, namely Gaussian, Gamma, Beta, and kernel densities. As a distance measure the Hellinger distance and the Kullback-Leibler divergence are considered. For the Gaussian, Gamma, and Beta models the closed-form expressions for evaluating the distance as a function of the model parameters are obtained. The advantages of the proposed quantification framework as compared to simple mean-based approaches are analyzed with numerical simulations. Two biological experiments are also considered. The first is the functional analysis of the p8 subunit of the TFIIH complex responsible for a rare hereditary multi-system disorder--trichothiodystrophy group A (TTD-A). In the second experiment the proposed methods are applied to assess the UV-induced DNA lesion repair rate. A good agreement between our in vivo results and those obtained with an alternative in vitro measurement is established. We believe that the computational simplicity and the effectiveness of the proposed quantification procedure will make it very attractive for different analysis tasks in functional proteomics, as well as in high-content screening.

Solution structure and self-association properties of the p8 TFIIH subunit responsible for trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare hereditary multi-system disorder associated with defects in nucleotide excision repair (NER) and transcription as consequences of mutations in XPB, XPD and p8/TTD-A subunits of transcription factor IIH (TFIIH). Here, we report the solution structure of the p8/TTD-A protein, a small alpha/beta protein built around an antiparallel beta-sheet that forms a homodimer with an extended interface. In order to characterize the dimer interface, we have introduced a mutation at position 44, which destabilizes the dimeric form of the protein. We have shown that this mutation has no effect on the intrinsic ability of p8/TTD-A to stimulate NER in vitro, but affects the capacity of p8/TTD-A to restore TFIIH concentration in TTD-A fibroblasts. Point mutations found in TTD-A patients are discussed on the basis of the present structure.

p8/TTD-A as a repair-specific TFIIH subunit.

How subunits of the transcription/repair factor TFIIH cooperate to allow for the removal of DNA lesions or for the transcription of genes is crucial to understand the functioning of this complex. Here, we reveal that p8/TTD-A, the tenth subunit of TFIIH, has a critical role in DNA repair where it triggers DNA opening by stimulating XPB ATPase activity together with the damage recognition factor XPC-hHR23B. Fluorescent antibody labeling shows that such opening is needed for the recruitment of XPA to the site of the damage. By contrast, p8 is dispensable for RNA synthesis and doesn't interfere with the transcriptional function of CAK, although both interact with the XPD subunit. Interestingly, p8 overexpression in TTD-XPD cells counteracts the detrimental effect of XPD mutations by restoring the cellular TFIIH concentration. These findings resolve the primary functions of p8 and unveil how TFIIH components specifically direct the complex toward repair or transcription.

Rejoining of radiation-induced DNA double-strand breaks: pulsed-field electrophoresis analysis of fragment size distributions after incubation for repair.

The repair of radiation-induced DNA double-strand breaks (DSBs) is frequently investigated by measuring the time-dependent decrease in the fraction of fragmented DNA that is able to enter electrophoresis gels. When transformed into equivalent doses without repair, such measurements are thought to reflect the removal of DSBs, and they typically exhibit a fast initial component and a decreasing rate at longer repair intervals. This formalism, however, assumes that the spatial distribution of unrejoined breakage resembles the pattern of induction of DSBs. While the size distributions for initial fragmentation, such as that resolved by conventional pulsed-field gel electrophoresis (PFGE) (between about 10(5) and 10(7) bp), are well known to agree with the prediction of random breakage, no data are available from studies explicitly testing this relationship for residual breakage. Therefore, Chinese hamster V79 cells and MeWo (human melanoma) cells were irradiated with different doses (10-100 Gy) or were incubated for repair for up to 4 h after a single dose of 100 Gy (V79) or 90 Gy (MeWo) before being subjected to PFGE. Fragment size distributions were calculated by convolution of the PFGE profiles with an appropriately generated size calibration function. The results clearly demonstrate an over-representation of smaller fragments (below about 2-3 Mbp) compared to the prediction of randomness for residual breakage. In consequence, the time-dependent decrease of dose-equivalent values calculated from data on the fraction released may not directly reflect DSB rejoining rates. The present findings are compatible with an earlier suggestion of slow rejoining of breaks which have been induced as multiple breaks (two or more) in large chromosomal loops, thus also predicting an increase of the slowly rejoining DSB fraction with increasing dose.