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Purpose: Preclinical studies indicate a normal tissue sparing effect when ultra-high dose rate (FLASH) radiation is used, while tumor response is maintained. This differential response has promising perspectives for improved clinical outcome. This study investigates tumor control and normal tissue toxicity of pencil beam scanning (PBS) proton FLASH in a mouse model. Methods and materials: Tumor bearing hind limbs of non-anaesthetized CDF1 mice were irradiated in a single fraction with a PBS proton beam using either conventional (CONV) dose rate (0.33–0.63 Gy/s field dose rate, 244 MeV) or FLASH (71–89 Gy/s field dose rate, 250 MeV). 162 mice with a C3H mouse mammary carcinoma subcutaneously implanted in the foot were irradiated with physical doses of 40–60 Gy (8–14 mice per dose point). The endpoints were tumor control (TC) assessed as no recurrent tumor at 90 days after treatment, the level of acute moist desquamation (MD) to the skin of the foot within 25 days post irradiation, and radiation induced fibrosis (RIF) within 24 weeks post irradiation.Results: TCD50 (dose for 50% tumor control) was similar for CONV and FLASH with values (and 95% confidence intervals) of 49.1 (47.0–51.4) Gy for CONV and 51.3 (48.6–54.2) Gy for FLASH. RIF analysis was restricted to mice with tumor control. Both endpoints showed distinct normal tissue sparing effect of proton FLASH with MDD50 (dose for 50% of mice displaying moist desquamation) of Conclusions: FLASH had the same tumor control as CONV, but reduced normal tissue damage assessed as acute skin damage and radiation induced fibrosis.

In human skin, keratinopoiesis is based on a functional hierarchy among keratinocytes, with rare slow-cycling stem cells responsible for the long-term maintenance of the tissue through their self-renewal potential, and more differentiated daughter progenitor cells actively cycling to permit epidermal renewal and turn-over every month. Skin is a radio-responsive tissue, developing all types of radiation damage and pathologies, including early tissue reactions such as dysplasia and denudation in epidermis, and later fibrosis in the dermis and acanthosis in epidermis, with the TGF-beta 1 pathway as a known master switch. Also there is a risk of basal cell carcinoma, which arises from epidermal keratinocytes, notably after oncogenic events in PTCH1 or TP53 genes. This review will cover the mechanisms of adverse human skin reactions and carcinogenesis after various types of exposures to ionizing radiation, with comparison with animal data when necessary, and will discuss the possible role of stem cells and their progeny in the development of these disorders. The main endpoints presented are basal cell intrinsic radiosensitivity, genomic stability, individual factors of risk, dose specific responses, major molecular pathways involved and the cellular origin of skin reactions and cancer. Although major advances have been obtained in recent years, the precise implications of epidermal stem cells and their progeny in these processes are not yet fully characterized.

Purpose: Radiotherapy using high linear energy transfer (LET) radiation is aimed at efficiently killing tumor cells while minimizing dose (biological effective) to normal tissues to prevent toxicity. It is well established that high LET radiation results in lower cell survival per absorbed dose than low LET radiation. However, whether various mechanisms involved in the development of normal tissue damage may be regulated differentially is not known. Therefore the aim of this study was to investigate whether two actions related to normal tissue toxicity, p53-induced apoptosis and expression of the profibrotic gene PAI-1 (plasminogen activator inhibitor 1), are differentially induced by high and low LET radiation.Methods and Materials: Cells were irradiated with high LET carbon ions or low LET photons. Cell survival assays were performed, profibrotic PAI-1 expression was monitored by quantitative polymerase chain reaction, and apoptosis was assayed by annexin V staining. Activation of p53 by phosphorylation at serine 315 and serine 37 was monitored by Western blotting. Transfections of plasmids expressing p53 mutated at serines 315 and 37 were used to test the requirement of these residues for apoptosis and expression of PAI-1.Results: As expected, cell survival was lower and induction of apoptosis was higher in high -LET irradiated cells. Interestingly, induction of the profibrotic PAI-1 gene was similar with high and low LET radiation. In agreement with this finding, phosphorylation of p53 at serine 315 involved in PAI-1 expression was similar with high and low LET radiation, whereas phosphorylation of p53 at serine 37, involved in apoptosis induction, was much higher after high LET irradiation.Conclusions: Our results indicate that diverse mechanisms involved in the development of normal tissue damage may be differentially affected by high and low LET radiation. This may have consequences for the development and manifestation of normal tissue damage. (C) 2012 Elsevier Inc.

Purpose: To assess in a rat model whether adding a subtolerance dose in a region adjacent to a high-dose irradiated subvolume of the parotid gland influences its response (bath-and-shower effect).Methods and Materials: Irradiation of the whole, cranial 50%, and/or the caudal 50% of the parotid glands of Wistar rats was performed using 150-MeV protons. To determine suitable (i.e., subtolerance) dose levels for a bath-dose, both whole parotid glands were irradiated with 5 to 25 Gy. Subsequently groups of Wistar rats received 30 Gy to the caudal 50% (shower) and 0 to 10 Gy to the cranial 50% (bath) of both parotid glands. Stimulated saliva flow rate (function) was measured before and up to 240 days after irradiation.Results : Irradiation of both glands up to a dose of 10 Gy did not result in late loss of function and is thus regarded subtolerance. Addition of a dose bath of I to 10 Gy to a high-dose in the caudal 50% of the glands resulted in enhanced function loss.Conclusion: Similar to the spinal cord, the parotid gland demonstrates a bath and shower effect, which may explain the less-than-expected sparing of function after IMRT. (C) 2009 Elsevier Inc.

Radionuclides are widely used in medicine. 131I is one of the most employed radionuclides and is administered to patients either bound to tumor targeting molecules or as halide to target the thyroid or thyroid cancer. 211At is proposed for radionuclide therapy and preclinical and clinical research on 211At-labeled tumor targeting molecules is on-going. The thyroid gland accumulates both 131I and 211At as halides and is an organ at risk. Additionally, 131I exposure of thyroid may occur from radioactive fallout from e.g. nuclear accidents. There is a lack of knowledge of molecular mechanisms in thyroid cells after 131I or 211At exposure. The overall aim of this work was to examine the transcriptomic and proteomic effects of 131I and 211At exposure on normal thyroid tissue in vivo. The influence of absorbed dose, dose-rate, time after administration, and radiation quality on gene expression regulation was studied. Another aim was to identify radiation-responsive genes in thyroid. Mice and rats were i.v. injected with 0.064-42 kBq 211At or 9-4700 kBq 131I. Resulting absorbed dose to thyroid from 211At and 131I exposures were 0.023-32 and 0.0058-34 Gy, respectively. Transcriptomic and proteomic responses in thyroid and plasma were measured 1-168 h after administration using RNA microarray and liquid chromatography mass spectrometry, respectively. Fold-change and adjusted p-value cut-offs of 1.5 and 0.01 were used to determine statistically significantly regulated transcripts. Pathway analyses were performed using Gene Ontology and the Ingenuity Pathway Analysis tool (p-value < 0.05). Plasma T4 and TSH levels were measured in rats using ELISA. The transcriptional response in thyroid tissue after 131I and 211At exposure varied with absorbed dose, dose-rate, time after administration, and radiation quality. In mice, 27 recurrently regulated genes were identified after 131I or 211At exposure and genes with similar function shared similar transcriptional regulation patterns. Additionally, regulation of several kallikrein genes was identified in mouse thyroid tissue after 131I or 211At administration. In rats, 2 recurrently genes were identified: Dbp and Slc47a2. Different biological functions were affected in response to different exposure conditions. For example, effects on immune response were found at 1, 6, and 168, but not 24 h after 1.7 kBq 211At administration in mice. An impact on rat thyroid function with regulation of 13 genes crucial for thyroid hormone synthesis was identified. The proteomic response to 32 Gy suggests hypoxia in thyroid and decreased thyroid function. Profound effects on gene expression regulation with distinct differences in response to different exposures were identified in mouse and rat thyroid tissue following 131I or 211At exposure. The transcriptional response likely depends to a varying degree on absorbed dose, dose-rate, time after administration, and radiation quality. Recurrently regulated genes were identified, and the biomarker applicability of these genes should be further assessed.

Purpose: To detect volume effects and possible regional differences in radiosensitivity of the rat parotid gland.Methods and Materials: Parotid glands of male albino Wistar rats were locally X-irradiated, with collimators with conformal radiation portals used to supply 100% volume and 50% cranial/caudal partial volumes. High-resolution magnetic resonance imaging was used to provide the outlines of the parotid glands. Single doses of up to 40 Gy were applied, and the effects on saliva secretion, measured with the aid of miniaturized Lashley cups, were followed up to 365 days after the irradiation.Results: Under conditions of equal mean absorbed doses and small variations in dose distribution, a pertinent volume effect was observed for late but not for early radiation damage. The late effects were different for the cranial part as compared with the caudal part of the parotid gland. The reduction in flow rate was much more severe after irradiation in the cranial part. After a single dose of 30 Gy, the reductions in flow rates were approximately 65% and 25% for the cranial and caudal parts, respectively. At that dose, no saliva flow was observed after irradiation of 100% of the gland.Conclusion: From the rat model studies presented, it is concluded that late radiation damage after partial irradiation of parotid glands shows region-dependent volume effects. This finding is expected to be relevant to the radiosensitivity of human salivary glands, and it implies that the predictive power of the mean dose concept in radiotherapeutic practice is limited. The finding of region-dependent late radiation damage also challenges the basic assumptions of most current normal tissue complication probability models for parotid gland function. (c) 2005 Elsevier Inc.

Purpose: To contribute to the understanding of the enigmatic radiosensitivity of the salivary glands by analysis of appropriate literature, especially with respect to mechanisms of action of early radiation damage, and to supply information on the possibilities of amelioration of radiation damage to the salivary glands after radiotherapy of head-and-neck cancer.Methods and Materials: Selected published data on the mechanism of salivary gland radiosensitivity and radioprotection were studied and analyzed.Results: From a classical point of view, the salivary glands should not respond as rapidly to radiation as they appear to do. Next to the suggestion of massive apoptosis, the leakage of granules and subsequent lysis of acinar cells was suggested to be responsible for the acute radiation-induced function loss of the salivary glands. The main problem with these hypotheses is that recently performed assays show no cell loss during the first days after irradiation, while saliva flow is dramatically diminished. The water secretion is selectively hampered during the first days after single-dose irradiation. Literature is discussed that shows that the compromised cells suffer selective radiation damage to the plasma membrane, disturbing signal transduction primarily affecting watery secretion. Although the cellular composition of the submandibular gland and the parotid gland are different, the damage response is very alike. The acute radiation-induced function loss in both salivary glands can be ameliorated by prophylactic treatment with specific receptor agonists.Conclusions: The most probable mechanism of action, explaining the enigmatic high radiosensitivity for early effects, is selective radiation damage to the plasma membrane of the secretory cells, disturbing muscarinic receptor stimulated watery secretion. Later damage is mainly due to classical mitotic cell death of progenitor cells, leading to a hampered replacement capacity of the gland for secretory cells, but is also caused by damage to the extracellular environment, preventing proper cell functioning. (c) 2005 Elsevier Inc.

Background Platinum compounds are potent anticancer drugs but also evoke considerable normal tissue damage. Here, we elucidate the molecular mechanisms contributing to the nephrotoxic effects of cisplatin. Methods We comparatively investigated the stress responses of rat kidney tubular (NRK-52E) and glomerular cells (RGE) following treatment with cisplatin (CisPt), oxaliplatin (OxaliPt) and carboplatin (CarboPt). To this end, cell viability, apoptosis, cell cycle progression, DNA damage response (DDR) and repair of DNA adducts were investigated. Results CisPt reduced the viability of tubular NRK-52E and glomerular RGE cells most efficiently. Cytotoxicity evoked by CarboPt occurred with a delay, which might be related to a retarded formation of Pt-(GpG) intrastrand crosslinks. RGE cells were more sensitive towards all platinum compounds than NRK-52E cells. Platinum drugs efficiently induced caspase-mediated apoptosis in tubular cells, while RGE cells favored G2/M arrest when treated with equitoxic platinum doses. Mitotic index of NKR-52E and RGE cells was worst affected by OxaliPt. Activation of the DDR was strikingly agent- and cell type-specific. Most comprehensive and substantial stimulation of DDR mechanisms was provoked by CisPt. Repair of Pt-(GpG) intrastrand crosslinks was best in RGE, which was reflected by high mRNA expression of nucleotide excision repair (NER) factors. Conclusions There are substantial differences regarding the cause of sensitivity and mechanisms of DDR between tubular and glomerular cells following platinum injury. CisPt is the most potent stimulator of the DDR. We hypothesize that specific DNA adducts and thereby forcefully activated pro-toxic DDR mechanisms contribute to the exceptionally high acute nephrotoxicity of CisPt.

The damage to normal pig bronchial mucosa caused by photodynamic therapy (PDT) using mTHPC and Photofrin as photosensitizers was evaluated. An endobronchial applicator was used to deliver the light with a linear diffuser and to measure the light fluence in situ. The applied fluences were varied, based on existing clinical protocols. A fluence finding experiment with short-term (1–2 days) response as an end point showed considerable damage to the mucosa with the use of Photofrin (fluences 50–275 J cm−2, drug dose 2 mg kg−1) with oedema and blood vessel damage as most important features. In the short-term mTHPC experiment the damage found was slight (fluences 12.5–50 J cm−2, drug dose 0.15 mg kg−1). For both sensitizers, atrophy and acute inflammation of the epithelium and the submucosal glands was observed. The damage was confined to the mucosa and submucosa leaving the cartilage intact. A long-term response experiment showed that fluences of 50 J cm−2 for mTHPC and 65 J cm−2 for Photofrin-treated animals caused damage that recovered within 14 days, with sporadic slight fibrosis and occasional inflammation of the submucosal glands. Limited data on the pharmacokinetics of mTHPC show that drug levels in the trachea are similar at 6 and 20 days post injection, indicating a broad time window for treatment. The importance of in situ light dosimetry was stressed by the inter-animal variations in fluence rate for comparable illumination conditions. © 1999 Cancer Research Campaign

There is compelling evidence that lymphocytes are a recurring feature in radiation damaged normal tissues, but assessing their functional significance has proven difficult. Contradictory roles have been postulated in both tissue pathogenesis and protection, although these are not necessarily mutually exclusive as the immune system can display what may seem to be opposing faces at any one time. While the exact role of T lymphocytes in irradiated normal tissue responses may still be obscure, their accumulation after tissue damage suggests they may be critical targets for radiotherapeutic intervention and worthy of further study. This is accentuated by recent findings that pathologically damaged “self,” such as occurs after exposure to ionizing radiation, can generate danger signals with the ability to activate pathways similar to those that activate adoptive immunity to pathogens. In addition, the demonstration of T cell subsets with their recognition radars tuned to “self” moieties has revolutionized our ideas on how all immune responses are controlled and regulated. New concepts of autoimmunity have resulted based on the dissociation of immune functions between different subsets of immune cells. It is becoming axiomatic that the immune system has the power to regulate radiation-induced tissue damage, from failure of regeneration to fibrosis, to acute and chronic late effects, and even to carcinogenesis. Our understanding of the interplay between T lymphocytes and radiation-damaged tissue may still be rudimentary but this is a good time to re-examine their potential roles, their radiobiological and microenvironmental influences, and the possibilities for therapeutic manipulation. This review will discuss the yin and yang of T cell responses within the context of radiation exposures, how they might drive or protect against normal tissue side effects and what we may be able do about it.

Purpose: To test the hypothesis that heart irradiation increases the risk of a symptomatic radiation-induced loss of lung function (SRILF) and that this can be well-described as a modulation of the functional reserve of the lung.Methods and Materials: Rats were irradiated with 150-MeV protons. Dose-response curves were obtained for a significant increase in breathing frequency after irradiation of 100%, 75%, 50%, or 25% of the total lung volume, either including or excluding the heart from the irradiation field. A significant increase in the mean respiratory rate after 6-12 weeks compared with 0-4 weeks was defined as SRILF, based on biweekly measurements of the respiratory rate. The critical volume (CV) model was used to describe the risk of SRILF. Fits were done using a maximum likelihood method. Consistency between model and data was tested using a previously developed goodness-of-fit test.Results: The CV model could be fitted consistently to the data for lung irradiation only. However, this fitted model failed to predict the data that also included heart irradiation. Even refitting the model to all data resulted in a significant difference between model and data. These results imply that, although the CV model describes the risk of SRILF when the heart is spared, the model needs to be modified to account for the impact of dose to the heart on the risk of SRILE Finally, a modified CV model is described that is consistent to all data.Conclusions: The detrimental effect of dose to the heart on the incidence of SRILF can be described by a dose dependent decrease in functional reserve of the lung. (c) 2007 Elsevier Inc.

The Ras-related C3 botulinum toxin substrate 1 (Rac1) belongs to the Ras-homologous (Rho) family of small GTPases, which transduce signals from the outside to the inside of a cell. Rac1 becomes activated upon ligand binding of a variety of receptors, including receptor tyrosine kinases and heterotrimeric G-protein-coupled receptors. After GTP loading by guanine exchange factors (GEFs), GTP-bound Rac1 engages numerous effector proteins, thereby eventually regulating cell motility and adhesion, cell cycle progression through G1, mitosis and meiosis, as well as cell death and metastasis.1 Besides, Rac1 adjusts cellular responses to genotoxic agents, such as UV light and alkylating agents, by rapid activation of stress kinases, including stress-activated protein kinases (SAPK/JNK) and p38 kinase, as well as transcription factors (e.g., AP-1, NFκB). Apart from regulating stress responses originating from the outer membrane, Rac1 may have additional nuclear functions. This assumption rests on the fact that Rac1 is not only present in the cytoplasm, but also in the nucleus, where it interacts with topoisomerase II (topo II),2 which is involved in the regulation of replication and transcription. Topo II isoforms are the major target of potent and widely used anticancer drugs, such as anthracyclines (e.g., doxorubicin) and podophyllotoxins (e.g., etoposide). Binding of these drugs to topo II causes inhibition of its DNA religation function, resulting in the formation of DNA double-strand breaks (DSBs). Pharmacological targeting of Rac1 by both HMG-CoA-reductase inhibitors (statins), small-molecule inhibitors, or clostridial toxins mitigates the DNA damage response (DDR) induced by topo II poisons.3 As the DDR is decision-making regarding activation of checkpoint control mechanisms, DNA repair, and/or apoptosis,4 it is conceivable that Rac1 holds a so-far-unappreciated nuclear function in response to genomic DNA damage. Since the Ras-homologous GTPase RhoA is also present in the nucleus, where it can be activated by ionizing radiation, it is possible that RhoA and Rac1 are integrated into the complex network of cellular responses to at least a subset of genotoxins by affecting both DNA damage-independent and DNA damage-dependent stress responses. Inhibition of Rac1 is a major mechanism of the well-known beneficial effect of statins in cardiovascular disease. Notably, statins also protect from cardiotoxicity evoked by anthracyclines in therapeutic relevant settings in vivo.5,6 Using Cre-based cardiac-specific knockout, inhibition of topo IIβ was recently suggested as the major cause of cardiotoxicity following anthracycline treatment.7 Hence, it is tempting to speculate that cardioprotection by statins rests on the inhibition of the Rac1-topo IIβ axis. This hypothesis, however, remains to be scrutinized by the analysis of cardiomyocyte-specific genetic knockouts of rac1. Analysis of the anthracycline response of mice lacking hepatic rac1 points to a double-edged function of Rac1 in acute and subacute settings.8 While rac1-deficient hepatocytes were protected from DNA damage induction and acute inflammatory and fibrotic responses following doxorubicin treatment, lack of rac1 promoted the subacute hepatotoxicity. Apparently, the influence of Rac1 signaling on the outcome of anthracycline exposure changes with time, illustrating the complexity of Rac1-regulated functions. Rac1 signaling enhances the interaction of topo II with its respective poisons, putatively by regulating the activity of protein kinases that phosphorylate topo II. In consequence, DSBs are formed, leading to the activation of the DDR. Rac1 also regulates inflammatory and fibrotic mechanisms by controlling the mRNA expression of cytokines, likely by regulating the activity of relevant transcription factors (e.g., AP-1, NFκB, STAT, Smad). Correspondingly, pharmacological or genetic abrogation of Rac1-regulated mechanisms disrupts stress responses stimulated by topo II poisons, resulting in beneficial effects in the short-term. On the other hand, Rac1 is required to alleviate the delayed harmful consequences of doxorubicin-induced injury by mitigating late inflammatory and fibrotic effects that emanate from anthracycline-induced cytotoxicity. The mechanisms underlying the dual biological activity of Rac1 in the context of anthracycline-induced stress responses, which comprise both beneficial and adverse effects, are still obscure. Furthermore, the influence of Rac1 on genotoxin-induced response seems to be agent-specific, as DNA damage and DDR evoked by ionizing radiation are not affected by Rac1.5,8 Apart from anticancer therapy-related aspects, there is another highly important issue that remains to be addressed. In vitro and in vivo data have demonstrated a key role of Rac1 in epidermal to mesenchymal transition (EMT) as well as Ras-mediated lung carcinogenesis. Whether Rac1 affects the acute and subacute response to chemical mutagens and, most importantly, also impacts chemical carcinogenesis is unknown. Thus, it would be of interest to elucidate, whether the mechanisms involved in tumor initiation and promotion are differently affected by Rac1 signaling. Moreover, Rac1 seems to interfere with aging as it regulates target of rapamycin (TOR), insulin secretion, and signaling, and also suppresses the expression of the senescence-associated cyclin-dependent kinase inhibitor p16.8 Taken together, targeting of Rac1 is proposed as a promising approach to interfere with manifold pathophysiological stress responses that favor DDR, inflammation, carcinogenesis, cell death, and aging. (Fig. 1) Figure 1.Model of Rac1-regulated response to genotoxic stress. Exposure to genotoxic agents rapidly stimulates membrane-bound receptors. In turn, guanine exchange factors (GEFs) for Rac1 become activated. GTP-bound active Rac1 couples to different ...