Multiparametric radiobiological assays show that variation of X-ray energy strongly impacts relative biological effectiveness: comparison between 220 kV and 4 MV

International audienceBased on classic clonogenic assay, it is accepted by the scientific community that, whatever the energy, the relative biological effectiveness of X-rays is equal to 1. However, although X-ray beams are widely used in diagnosis, interventional medicine and radiotherapy, comparisons of their energies are scarce. We therefore assessed in vitro the effects of low- and high-energy X-rays using Human umbilical vein endothelial cells (HUVECs) by performing clonogenic assay, measuring viability/mortality, counting γ-H2AX foci, studying cell proliferation and cellular senescence by flow cytometry and by performing gene analysis on custom arrays. Taken together, excepted for γ-H2AX foci counts, these experiments systematically show more adverse effects of high energy X-rays, while the relative biological effectiveness of photons is around 1, whatever the quality of the X-ray beam. These results strongly suggest that multiparametric analysis should be considered in support of clonogenic assay

The TGF-β/Smad Repressor TG-Interacting Factor 1 (TGIF1) Plays a Role in Radiation-Induced Intestinal Injury Independently of a Smad Signaling Pathway

Despite advances in radiation delivery protocols, exposure of normal tissues during the course of radiation therapy remains a limiting factor of cancer treatment. If the canonical TGF-β/Smad pathway has been extensively studied and implicated in the development of radiation damage in various organs, the precise modalities of its activation following radiation exposure remain elusive. In the present study, we hypothesized that TGF-β1 signaling and target genes expression may depend on radiation-induced modifications in Smad transcriptional co-repressors/inhibitors expressions (TGIF1, SnoN, Ski and Smad7). In endothelial cells (HUVECs) and in a model of experimental radiation enteropathy in mice, radiation exposure increases expression of TGF-β/Smad pathway and of its target gene PAI-1, together with the overexpression of Smad co-repressor TGIF1. In mice, TGIF1 deficiency is not associated with changes in the expression of radiation-induced TGF-β pathway-related transcripts following localized small intestinal irradiation. In HUVECs, TGIF1 overexpression or silencing has no influence either on the radiation-induced Smad activation or the Smad3-dependent PAI-1 overexpression. However, TGIF1 genetic deficiency sensitizes mice to radiation-induced intestinal damage after total body or localized small intestinal radiation exposure, demonstrating that TGIF1 plays a role in radiation-induced intestinal injury. In conclusion, the TGF-β/Smad co-repressor TGIF1 plays a role in radiation-induced normal tissue damage by a Smad-independent mechanism

Preclinical model of stereotactic ablative lung irradiation using arc delivery in the mouse:effect of beam size changes and dose effect at constant collimation

International audiencePurpose: Stereotactic body radiation therapy is a therapeutic option offered to high surgical risk cancer patients with lung cancer. Focal lung irradiation in mice is a new preclinical model to help understand the development of lung damage in this context. Here we developed a mouse model of lung stereotactic therapy using arc delivery and monitored the development of lung damage while varying beam size and dose delivered.Methods and Materials: C57BL/6JRj mice were exposed to 90 Gy focal irradiation on the left lung, using 1 mm diameter, 3 x 3 mm2, 7 x 7 mm2 or 10 x 10 mm2 beam collimation for beam size effect, and using 3 x 3 mm2 beam collimation delivering 20 to 120 Gy for dose effect. Long-term lung damage was monitored with micro-CT imaging together with anatomopathological and gene expression measurements in the injured patch and the ipsilateral and contralateral lungs. Results: Both 1 mm diameter and 3 x 3 mm2 beam collimation allow long-term studies, but only 3 mm beam collimation generates lung fibrosis when delivering 90 Gy. Dose-effect studies with constant 3 mm beam collimation revealed a dose of 60 Gy as the minimum to obtain lung fibrosis 6 months post-exposure. Lung fibrosis development was associated with club cell depletion and increased type II pneumocyte numbers. Lung injury developed with ipsilateral and contralateral consequences such as parenchymal thickening and gene expression modifications. Conclusions: Arc therapy allows long-term studies and dose escalation without lethality. In our dose-delivering conditions, dose-effect studies revealed that 3 x 3 mm2 beam collimation to a minimum single dose of 60 Gy enables preclinical models for the assessment of lung injury within a 6-month period. This model of lung tissue fibrosis in a time length compatible with mouse life span may offer good prospects for future mechanistic studies

Pravastatin Limits Radiation-Induced Vascular Dysfunction in the Skin

About half of people with cancer are treated with radiation therapy; however, normal tissue toxicity still remains a dose-limiting factor for this treatment. The skin response to ionizing radiation may involve multiple inflammatory outbreaks. The endothelium is known to play a critical role in radiation-induced vascular injury. Furthermore, endothelial dysfunction reflects a decreased availability of nitric oxide. Statins have been reported to preserve endothelial function through their antioxidant and anti-inflammatory activities. In this study, wild type and endothelial nitric oxide synthase (eNOS)-/- mice were subjected to dorsal skin irradiation and treated with pravastatin for 28 days. We demonstrated that pravastatin has a therapeutic effect on skin lesions and abolishes radiation-induced vascular functional activation by decreasing interactions between leukocytes and endothelium. Pravastatin limits the radiation-induced increase of blood CCL2 and CXCL1 production expression of inflammatory adhesion molecules such as E-selectin and intercellular adhesion molecule-1, and inflammatory cell migration in tissues. Pravastatin limits the in vivo and in vitro radiation-induced downregulation of eNOS. Moreover, pravastatin has no effect in eNOS-/- mice, demonstrating that eNOS plays a key role in the beneficial effect of pravastatin in radiation-induced skin lesions. In conclusion, pravastatin may be a good therapeutic approach to prevent or reduce radiation-induced skin damage

Preclinical model of stereotactic ablative lung irradiation using arc delivery in the mouse:fractionation effect

International audiencePurpose: pre-clinical modelling of radiation-induced lung damage is being challenged by emerging radiation therapies, such as stereotactic body radiation therapy. The aim of the present study was to implement previously published data on focal lung irradiation in mice using different fractionation schedules, with the objective of developing lung fibrosis in a time lapse that is compatible with the lifespan of a rodent.Methods and materials: the left lungs of C57BL/6JRj mice were exposed to ionizing radiation using arc therapy and 3 x 3 mm beam collimation. Three-fraction schedules delivered in 1 week were used with 20, 28, 40 and 50 Gy doses per fraction. Lung tissue opacification, global histological damage and the numbers of type II pneumocytes and club cells were assessed 6 months post-exposure, together with the gene expression of several lung cells and inflammation markers.Results: administration of 3 x 40 or 3 x 50 Gy generated focal lung fibrosis after 6 months, with tissue opacification visible by cone beam computed tomography, tissue scarring and consolidation, decreased club cell numbers and a reactive increase in the number of type II pneumocytes.Conclusions: a fractionation schedule using an arc-therapy-delivered 3 fraction/1 week regimen with 3 x 3 mm beam collimation was complex but feasible, with satisfying image-guided animal repositioning. Such a schedule generates focal lung fibrosis within 6 months when using 40 or 50 Gy per fraction. A comparison with previously published laboratory data suggests that, in this focal lung irradiation configuration, administrating a Biological Effective Dose ≥ 1000 Gy should be recommended to obtain lung fibrosis within 6 months

Variation of 4 MV X-ray dose rate in fractionated irradiation strongly impacts biological endothelial cell response in vitro

International audienceComparisons of X-ray beam dose rates are scarce although these beams are widely used in medical diagnosis or radiotherapy. We have recently demonstrated in vitro and in vivo, that for a single dose of irradiation, the relative biological effectiveness (RBE) deviates from 1 when changing the dose rate of high energy X-ray beams. To further investigate the impact of the dose rate on RBE, in this study we performed in vitro fractionated irradiations by using the same two dose rates (0.63 and 2.5 Gy.min-1) of high-energy X-rays (both at 4 MV) on normal endothelial cells (HUVECs). We studied the viability/mortality, measured cellular senescence by flow cytometry and performed gene analysis on custom arrays. Taken together, these experiments show that the RBE of photons deviates from 1 when varying the dose rate of high-energy X-rays in fractionated irradiations. These results strengthen the interest of multiparametric analysis approaches in providing an accurate evaluation of the outcomes of irradiated cells in support of clonogenic assays, especially when such assays are not feasible

Ultratrace LC-MS/MS Analysis of Segmented Calf Hair for Retrospective Assessment of Time of Clenbuterol Administration in Agriforensics

In agriforensics, time of administration is often debated when illegal drug residues, such as clenbuterol, are found in frequently traded cattle. In this proof-of-concept work, the feasibility of obtaining retrospective timeline information from segmented calf tail hair analyses has been studied. First, an ultraperformance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) hair analysis method was adapted to accommodate smaller sample sizes and in-house validated. Then, longitudinal 1 cm segments of calf tail hair were analyzed to obtain clenbuterol concentration profiles. The profiles found were in good agreement with calculated, theoretical positions of the clenbuterol residues along the hair. Following assessment of the average growth rate of calf tail hair, time of clenbuterol administration could be retrospectively determined from segmented hair analysis data. The data from the initial animal treatment study (n = 2) suggest that time of treatment can be retrospectively estimated with an error of 3–17 days

Variation of 4 MV X-ray dose rate strongly impacts biological response both in vitro and in vivo

International audienceBased on classic clonogenic assay, it is accepted by the scientific community that, whatever the energy or the dose rate, the relative biological effectiveness of X-rays is equal to 1. However, although X-ray beams are widely used in diagnosis, interventional medicine and radiotherapy, comparisons of their dose rates are scarce. We therefore assessed in vitro the effects of high-energy X-rays at two dose rates (0.63 and 2.5 Gy/min) using normal endothelial cells (HUVECs) by using clonogenic assay, measuring viability/mortality, studying the cell cycle and cellular senescence by flow cytometry and by performing gene analysis on custom arrays. In order to consolidate these data, we performed localized irradiation of exteriorized small intestine at 0.63 and 2.5 Gy/min. Interestingly, in vivo validation has shown a significantly higher loss of weight at the higher dose when irradiating to 19 Gy a small fragment of exteriorized small intestine of C57Bl6J mice. Nevertheless, no significant differences were observed in lesioned scores between the two dose rates, while bordering epithelium staining indicated twofold greater severe damage at 2.5 Gy/min compared to 0.63 Gy/min at one week post-irradiation. Taken together, these experiments systematically show more adverse effects of high energy X-rays at 2.5 Gy/min, while the relative biological effectiveness of photons is around 1, whatever the quality of the X-ray beam. These results strongly suggest that multiparametric analysis should be considered in support of clonogenic assay

Mesenchymal stem cells limit vascular and epithelial damage and restore the impermeability of the urothelium in chronic radiation cystitis

International audienceAbstract Background Cellular therapy seems to be an innovative therapeutic alternative for which mesenchymal stem cells (MSCs) have been shown to be effective for interstitial and hemorrhagic cystitis. However, the action of MSCs on chronic radiation cystitis (CRC) remains to be demonstrated. The aim of this study was to set up a rat model of CRC and to evaluate the efficacy of MSCs and their mode of action. Methods CRC was induced by single-dose localized irradiation of the whole bladder using two beams guided by tomography in female Sprague–Dawley rat. A dose range of 20–80 Gy with follow-up 3–12 months after irradiation was used to characterize the dose effect and the kinetics of radiation cystitis in rats. For the treatment, the dose of 40 Gy was retained, and in order to potentiate the effect of the MSCs, MSCs were isolated from adipose tissue. After expansion, they were injected intravenously during the pre-chronic phase. Three injections of 5 million MSCs were administered every fortnight. Follow-up was performed for 12 months after irradiation. Results We observed that the intensity and frequency of hematuria are proportional to the irradiation dose, with a threshold at 40 Gy and the appearance of bleeding from 100 days post-irradiation. The MSCs reduced vascular damage as well as damage to the bladder epithelium. Conclusions These results are in favor of MSCs acting to limit progression of the chronic phase of radiation cystitis. MSC treatment may afford real hope for all patients suffering from chronic radiation cystitis resistant to conventional treatments