John Gueulette

Citrulline: A physiologic marker enabling quantitation and monitoring of epithelial radiation-induced small bowel damage

PURPOSE Small bowel irradiation results in epithelial cell loss and consequently impairs function and metabolism. We investigated whether citrulline, a metabolic end product of small bowel enterocytes, can be used for quantifying radiation-induced epithelial cell loss. METHODS AND MATERIALS NMRI mice were subjected to single-dose whole body irradiation (WBI). The time course of citrullinemia was assessed up to 11 days after WBI. A dose-response relationship was determined at 84 h after WBI. In addition, citrullinemia was correlated with morphologic parameters at this time point and used to calculate the dose-modifying factor (DMF) of glutamine and amifostine on acute small bowel radiation damage. RESULTS After WBI, a time- and dose-dependent decrease in plasma citrulline level was observed with a significant dose-response relationship at 84 h. At this time point, citrullinemia significantly correlated with jejunal crypt regeneration (p < 0.001) and epithelial surface lining (p = 0.001). A DMF of 1.0 and 1.5 was computed at the effective dose 50 (ED50) level for glutamine and amifostine, respectively. CONCLUSIONS Citrullinemia can be used to quantify acute small bowel epithelial radiation damage after single-dose WBI. Radiation-induced changes in citrullinemia are most pronounced at 3 1/2 to 4 days postirradiation. At this time point, citrullinemia correlates with morphologic endpoints for epithelial radiation damage.

Life-shortening and Disease Incidence in C57bi Mice After Single and Fractionated-gamma and High-energy Neutron Exposure

C57Bl Cnb mice were exposed to single or fractionated d(50)+Be neutrons or 137Cs gamma-ray exposure at 12 weeks of age and were followed for life-shortening and disease incidence. The data were analyzed by the Kaplan-Meier procedure using as criteria cause of death and possible cause of death. Individual groups were compared by a modified Wilcoxon test according to Hoel and Walburg, and entire sets of different doses from one radiation schedule were evaluated by the procedure of Peto and by the Cox proportional hazard model. No significant difference was found in life-shortening of C57Bl mice between a single gamma and neutron exposure. Gamma fractionation was clearly less effective in reducing survival time than a single exposure. On the contrary, fractionation of neutrons was slightly although not significantly more effective in reducing life span than a single exposure. Life-shortening appeared to be a linear function of dose in all groups studied. The data on causes of death show that malignant tumors, particularly leukemias including thymic lymphoma, and noncancerous late degenerative changes in lung were the principal cause of life-shortening after a high single gamma exposure. Exposure delivered in 8 fractions 3 h apart was more effective in causing leukemias and all carcinomas and sarcomas than one delivered in 10 fractions 24 h apart or in a single session. Following a single neutron exposure, leukemias and all carcinomas and sarcomas appeared to increase somewhat more rapidly with dose than after gamma irradiation. No significant difference in the incidence of leukemias and all carcinomas and sarcomas was noted between a single and a fractionated neutron exposure.

Early Recovery for Intestinal Stem Cells, as a Function of Dose per Fraction, Evaluated by Survival Rate After Fractionated Irradiation of the Abdomen of Mice'

times (N X D8 or N X 2Di), the number of fractions N being adapted to their size in order to reach a suitable rate of survival. The overall treatment time for each compared protocol was kept constant. Intervals i between the two subfractions 2DA of 3.5 and 24 hr were studied. No significant difference appeared in the variation of Dr as a function of D, related to interval i for the two considered intervals.

RBE variation as a function of depth in the 200-MeV proton beam produced at the National Accelerator Centre in Faure (South Africa).

BACKGROUND AND PURPOSE Thorough knowledge of the RBE of clinical proton beams is indispensable for exploiting their full ballistic advantage. Therefore, the RBE of the 200-MeV clinical proton beam produced at the National Accelerator Centre of Faure (South Africa) was measured at different critical points of the depth-dose distribution. MATERIAL AND METHODS RBEs were determined at the initial plateau of the unmodulated and modulated beam (depth in Perspex = 43.5 mm), and at the beginning, middle and end of a 7-cm spread-out Bragg peak (SOBP) (depths in Perspex = 144.5, 165.5 and 191.5 mm, respectively). The biological system was the regeneration of intestinal crypts in mice after irradiation with a single fraction. RESULTS Using 60Co gamma-rays as the reference, the RBE values (for a gamma-dose of 14.38 Gy corresponding to 10 regenerated crypts) were found equal to 1.16 +/- 0.04, 1.10 +/- 0.03, 1.18 +/- 0.04, 1.12 +/- 0.03 and 1.23 +/- 0.03, respectively. At all depths, RBEs were found to increase slightly (about 4%) with decreasing dose, in the investigated dose range (12-17 Gy). No significant RBE variation with depth was observed, although RBEs in the SOBP were found to average a higher value (1.18 +/- 0.06) than in the entrance plateau (1.13 +/- 0.04). CONCLUSION An RBE value slightly larger than the current value of 1.10 should be adopted for clinical application with a 200-MeV proton beam.

In vivo colocalization of 2-nitroimidazole EF5 fluorescence intensity and electron paramagnetic resonance oximetry in mouse tumors.

BACKGROUND AND PURPOSE The primary objective of this study was to establish in vivo the relationship between 2-2-nitro-1H-imidazol-1yl-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5) adduct formation and intratumoral oxygen concentrations measured by electron paramagnetic resonance (EPR) in a tumor model mimicking a clinical situation. The secondary objective was an attempt to calibrate in situ the immunofluorescence (IF) signal with EPR oximetry. MATERIALS AND METHODS IM syngeneic fibrosarcoma (NFSA) bearing C3H mice were used. Three days after injection of a paramagnetic charcoal into the tumor, the mice were anesthetized, injected with the hypoxic marker EF5, and monitored every 20 min for 3 h with a low-frequency EPR spectrometer. Animals were allowed to breath either under 21 or 100% O(2). Tumors were then harvested, frozen, cut into sections including the charcoal and processed for EF5 adducts detection using monoclonal antibodies. Slices were viewed with a fluorescence microscope and 190x140 micrometer areas surrounding the charcoal were digitized and analyzed with the NIH-Image and Adobe Photoshop software. The fluorescence intensity (FI) was measured in the whole pictures and in strips of 10 micrometer around the charcoal. RESULTS EF5 binding increased with decreasing pO(2), most substantially at pO(2) below 5 mm Hg. Baseline (ambient air) pO(2) reached 3.2+/-2.1 mm Hg in NFSA tumors. It increased to 9.8+/-3.2 mm Hg under 100% O(2). A statistically significant correlation was observed on an individual tumor basis between the FI in the first 10 micrometer strip around the charcoal and the pO(2) determined by EPR oximetry (Wilcoxon signed rank test: P<0.001). CONCLUSIONS The present study confirms the intrinsic relationship between EF5 adduct binding and intratumoral pO(2) in an in vivo environment under biologically-relevant pO(2) values of less than 10 mm Hg.

Caspase 8-mediated cleavage of the pro-apoptotic BCL-2 family member BID in p53-dependent apoptosis

The objective of this study was to characterize the apoptotic pathways activated by fast neutrons in the human lymphoblastoid cell line TK6 and in its p53 -/- derivative. Our results demonstrate that while p53 is not required for neutron-induced apoptosis, as previously shown, it does affect the kinetics of apoptosis and the molecular pathways leading to the activation of effector caspases. Indeed, rapid p53-dependent apoptosis was associated with the activation of caspase 9, 8, 3, and 7 and the cleavage of BID by caspase 8. In contrast, the slow-occurring p53-independent apoptotic process, mediated by caspase 7, took place without BID cleavage and loss of transmembrane mitochondrial potential. Altogether, our findings highlight an essential role for caspase 8-mediated BID cleavage, in the course of p53-dependent apoptosis triggered by fast neutrons in lymphoid cells. They also demonstrate that this mechanism is not involved in p53-independent apoptosis.

Measurements of radiobiological effectiveness in the 85 MeV proton beam produced at the cyclotron CYCLONE of Louvain-la-Neuve, Belgium.

The RBE of the 85 MeV proton beam produced at the cyclotron of Louvain-la-Neuve using 60Co gamma rays as the reference radiation was determined for survival of Chinese hamster ovary cells in vitro and for intestinal crypt regeneration in mice in vivo. Cell survival curves determined at different depths yielded, for a surviving fraction (SF) of 0.01, RBE values of 1.11 +/- 0.05 at the initial plateau of the unmodulated beam, 1.10 +/- 0.03 at the middle of a 0.5-cm spread-out Bragg peak (SOBP), 1.03 +/- 0.03 at the beginning of a 3-cm SOBP and 1.07 +/- 0.03 at the end of a 3-cm SOBP. The highest RBE values were obtained at the middle of the 0.5-cm SOBP and at the end of the 3-cm SOBP (RBE = 1.22 and 1.16, respectively, at SF = 0.5), although the variations are not statistically significant. Irradiations with 3-Gy fractions separated by an interval of 3.5 h yielded RBEs of 1.11 +/- 0.30 and 0.90 +/- 0.32 at the initial plateau and at the middle of the 0.5-cm SOBP, respectively. Irradiations of mice at the middle of the 3-cm SOBP yielded an RBE of 1.08 +/- 0.03 for 20 regenerated crypts at a proton dose of 12.3 Gy.

High-LET radiation combined with oxaliplatin induce autophagy in U-87 glioblastoma cells

Modern protocols of concomitant chemo/radiotherapy provide a very effective strategy to treat certain types of tumors. High-linear energy transfer (LET) radiations, on the other hand, have an increased efficacy against cancer with low radiosensibility and critical localization. We previously reported that oxaliplatin, a third generation platinum drug, was able to reinforce the cytotoxicity of an irradiation by fast neutrons towards human glioblastoma U-87 cells in culture. We show here that such a combination has the capacity to enhance the number of double strand breaks in DNA and to induce autophagy in these cells. Xenografts experiments were further performed in nude mice subcutaneously transplanted with U-87 cells. When injected shortly before a single irradiation by fast neutrons, oxaliplatin causes a marked reduction of tumor growth compared with the irradiation alone. Overall, our data indicate the unique cytotoxic mechanism of a combined high-LET irradiation and oxaliplatin treatment modality and suggest its potential application in anticancer therapy.

Intestinal crypt regeneration in mice : a biological system for quality assurance in non-conventional radiation therapy

BACKGROUND AND PURPOSE The Relative Biological Effectiveness (RBE) of 8 fast-neutron beams, 5 proton beams and 1 carbonion beam was determined using as biological criterion intestinal crypt regeneration in mice, i.e. an in vivo system. These beams are used or planned for clinical cancer therapy applications. In addition, the RBE of 6 epithermal neutron beams, used or planned for Boron Neutron Capture Therapy (BNCT), was determined; no boron was administered. The goal of the program was to improve the exchange of information between the centers, facilitate the interpretation of the results and increase the safety of the clinical applications. MATERIALS AND METHODS In all visited centers, the same technique was applied in the same conditions by the same radiobiology team. The number of regenerating crypts per circumference was scored 3.5 days after single fraction total body irradiation. The control irradiations were performed locally using cobalt-60 units. The mice were randomized according to radiation quality and dose level. RESULTS (1) For fast neutron beams, the RBE (Ref. cobalt-60 gamma rays) increases with decreasing energy (from approximately 1.7 for p(65)+Be neutrons to approximately 2.4 for d(14.5)+Be neutrons). In addition, it is specific to each facility and depends on the nuclear reaction (p or d + Be), target and collimation type. (2) For proton beams, the RBEs (Ref cobalt-60 gamma rays) at the reference position (middle of a 7-cm Spread Out Bragg Peak, SOBP) range between 1.08 and 1.18. They might differ by approximately 6-8% according to the mode of beam production or delivery. The RBEs at the end of the SOBP are always 5-10 % higher than at the middle of the SOBP. (3) For the carbon ion beam studied at NIRS in Chiba, Japan, the RBE significantly increases with depth. Relative to gamma rays, it ranges from 1.3 in the initial plateau, 1.6 at the beginning, 1.7 at the middle and 1.9 at the end of a 6-cm SOBP. 4) In BNCT beams, the radiation quality (in particular the relative contribution of the different dose components) varies rapidly with depth and depends strongly on the arrangement of the irradiation set-up (e.g. presence or not of back scattering material). Moreover, the (total) dose rates are highly variable (from 0.05 to approximately 0.5 Gy/min) according to the power of the reactors. Wide range of RBE values (Ref. gamma rays) was thus obtained (RBE = 1.4 - 2.2) at shallow depths of 1.5 - 2.5 cm. DISCUSSION AND CONCLUSION Intestinal crypt regeneration in mice is an in vivo system perfectly suitable to perform intercomparisons between centers applying different types of non-conventional radiation qualities. It was proven to be reproducible, reliable and accurate, and becomes progressively recognized worldwide as part of the Quality Control (QA) procedures for new beams. It should be stressed that the observed RBE for intestinal crypt cells after a single high dose provide some radiobiological characterization of the radiation quality but cannot be used as the RBE weighting factor in clinical prescriptions.

Radiobiological rationale and patient selection for high-LET radiation in cancer therapy.

The rationale for introducing ion beams in cancer therapy is the high level of physical selectivity that can be achieved with ions, equal or even better than with proton beams or modern photon techniques, as well as the potential advantage of high-LET radiations for some tumour types and sites. The radiobiological arguments for high-LET radiation in cancer therapy are reviewed: reduction of OER in the case of hypoxic and poorly-reoxygenating tumours, and the lesser importance of repair phenomena which are a problem in controlling repair-proficient photon-resistant tumours. Fast neutrons were the first type of high-LET radiation used clinically, and were often applied under suboptimal technical conditions. Nevertheless, useful clinical information was derived from the neutron experience. A greater benefit from neutrons than from conventional radiotherapy was found for several tumour sites. The present discussion is limited to the results for salivary gland tumours and prostatic adenocarcinoma. Based on the fast neutron experience, radiobiological arguments, and the added benefit of excellent physical selectivity of ion beams, the potential clinical indications for high-LET ions are discussed: hypoxic, slowly growing and well-differentiated photon-resistant tumours. One of the main remaining issues is the selection of individual patients for high- or low-LET radiation. Since the physical selectivity of ions now matches that obtained with other techniques, the selection of patients will be based only on the radiobiological characteristics of the tumour.