Background and purposePreclinical evidence suggesting gemcitabine potentiates the anti-tumor effects of irradiation has resulted in clinical trials to evaluate the treatment efficacy of gemcitabine and concurrent thoracic irradiation in non-small-cell lung cancer (NSCLC). Although these studies demonstrated favorable tumor response, this combined treatment modality was accompanied by severe treatment-related toxicities predominantly of the lung. In an attempt to elucidate the determinants of lung toxicity for gemcitabine, we analyzed the expression of the pro-inflammatory cytokines TNF-α, IL-1α and IL-6 in the lung tissue of mice treated with gemcitabine and concurrent thoracic irradiation.Materials and methodsFour study groups were defined: C57BL/6J mice that received neither irradiation nor gemcitabine (NT-group), those that received gemcitabine (120 mg/kg intraperitoneal, i.p.) but no irradiation (GEM-group), those that underwent thoracic irradiation (12Gy) without gemcitabine (XRT-group), and those that received both gemcitabine (120 mg/kg i.p., 2 h before irradiation) and thoracic irradiation (GEM/XRT-group). The mice were sacrificed at 1 h, 1 and 3 days, 1, 2 and 4 weeks post-treatment (p.t.). The mRNA expression of TNF-α, IL-1α and IL-6 in the lung tissue was quantified by competitive RT-PCR. The cellular origin of the cytokine expression was identified by immunohistochemistry. The cytokine expression was correlated with histopathological alterations.ResultsThe TNF-α, IL-1α and IL-6 expression in the lung tissue of the GEM/XRT mice was clearly higher at all assessment time points compared to the NT mice (statistically significant at 1 h, 1 and 3 days, 1, 2 and 4 weeks p.t.), XRT mice (statistically significant at 1 week p.t.) or GEM mice (statistically significant at 1 h, 1 and 2 weeks p.t.). Maximal treatment-induced cytokine expression in the lung tissue of the GEM/XRT mice occurred already at 1 week p.t. (TNF-α: 30.9±5.3/IL-1α: 28.3±5.0/IL-6: 4.9±0.1 times basal level), and coincides with pathohistologically discernable interstitial pneumonitis. The elevated levels of TNF-α and IL-1α have been found to correlate with immunohistochemical staining of the bronchiolar epithelium and predominantly of inflammatory cells.ConclusionsOur data provide evidence that the increased expression of pro-inflammatory cytokines and the induction of a cytokine-triggered inflammatory response may be a determinant of the observed elevated lung toxicity after concurrent treatment with gemcitabine and thoracic irradiation.

The lung is the major dose-limiting organ for radiotherapy of cancer in the thoracic region. The dysregulation of a variety of inflammatory and fibrogenic cytokines has been shown to play an important role in the pathogenesis of radiation-induced lung injury. The present work is a detailed study on the temporal and spatial release of the pro-inflammatory cytokines TNF-alpha, IL-1 alpha and IL-6 in the lung tissue of C57BL/6 mice after thoracic irradiation with 12 Gy. C57BL/6J mice were exposed to either sham irradiation or single fraction of 12 Gy delivered to the thorax. Treated and sham-irradiated control mice were sacrificed at 0.5 hour (h), 1h, 3h, 6h, 12h, 24h, 48h, 72h, 1 week (w), 2w, 4w, 8w, 16w, 24w post-irradiation (p.i.). Real-time multiplex RT-PCR was established to evaluate the mRNA expression of TNF-alpha, IL-1 alpha and IL-6 in the lung tissue of the mice. Immunohistochemical detection methods (APAAP, ABC) and automated image analysis were used to quantify the protein expression of TNF-alpha, IL-1 alpha and IL-6 (percentage of positively stained area). Radiation-induced release of the pro-inflammatory cytokines TNF-alpha, IL-1 alpha and IL-6 in the lung tissue was detectable within the first hours after thoracic irradiation. We observed a statistically significant up-regulation for TNF-alpha at 1h p.i. on mRNA- and at 6h p.i. on protein level (7.2 ± 0.02 %), for IL-1 alpha at 6h p.i. on mRNA- and at 12h p.i. on protein level (27.6 ± 0.11 %), for IL-6 at 6h p.i. on mRNA- and at 12h p.i. on protein level (7.1 ± 0.02 %). By means of immunohistochemistry, we can clearly demonstrate that the bronchiolar epithelium is the most prominent source of these inflammatory cytokines in the first hours after lung irradiation. During the stage of acute pneumonitis, the bronchiolar epithelium as well as inflammatory cells in the lung parenchyma produce high amounts of TNF-alpha (with the maximal value at 4w p.i.: 9.5 ± 0.02 %), IL-1 alpha (with the peak value at 8w p.i.: 14.8 ± 0.1 %) and IL-6 (with the peak value at 8w p.i.: 4.3 ± 0.01 %). In the present study we can clearly demonstrate the immediate expression of the pro-inflammatory cytokines TNF-alpha, IL-1 alpha and IL-6 in the bronchiolar epithelium in the first hours after lung irradiation. A second long lasting release of these cytokines by the bronchiolar epithelium and by inflammatory cells was observed at the onset of acute pneumonitis. Therefore, we postulate that lung irradiation causes immediate epithelial injury with the bronchiolar epithelium becoming a significant source of pro-inflammatory cytokines capable to promote inflammation through recruitment and activation of inflammatory cells

Purpose : The shape of the dose-effect curve for neutrons, i.e. the question as to whether the curve is linear or supralinear in the low-dose region, is still not clear. Therefore, the mutagenic effect of very low doses of low-energy neutrons was determined.Materials and methods : Human-hamster hybrid A(L) cells contain human chromosome 11, which expresses the membrane protein CD59. This membrane protein can be detected immunologically and quantified by flow cytometry. The A L cells were irradiated with neutrons of 0.565, 2.5 or 14.8 MeV and the results were compared with those after 200 kVp X-rays. Before irradiation, cells spontaneously mutated in the CD59 gene were removed by magnetic cell sorting (MACS).Results : The relative biological effectiveness (RBE) for CD59 mutation induction was 19.8 (+/-2.7) for 0.565 MeV, 10.2 (+/-1.9) for 2.5 MeV, and 10.2 (+/-1.6) for 14.8 MeV neutrons. Linear mutation responses were obtained with all radiations except for 14.8 MeV neutrons where a supralinear curve may be a better fit. The deletion spectrum of mutated cell clones showed 29 Mbp deletions on average after irradiation with 0.069 Gy of 0.565 MeV neutrons. This scale of deletions is similar to that after 3 Gy 100 kV X-rays (=34 Mbp). For 50% cell survival, the RBE of the neutrons was 11 compared with 200 kV X-rays.Conclusion : Neutrons of low energies (0.565 or 2.5 MeV) produce a linear dose-response for mutation in the tested dose range of 0.015-0.15 Gy. The neutron curve of 14.8 MeV can be approximated by a curvilinear or linear function.

Purpose : To measure the mutagenic effectiveness of low-filtered 30 kVp X-rays, mammography X-rays and conventional (200 kVp) X-rays in mammalian cells. Materials and methods : Two different cell lines and mutation assays were used. Exponentially growing SV40-transformed human fibroblasts were exposed to graded doses of mammography (29 kVp, tungsten anode, 50 μ m Rh filter) or conventional X-rays and the frequency of 6-thioguanine-resistent HPRT-deficient mutants was determined. Exponentially growing hamster A L cells, which contain a single human chromosome 11 conferring the expression of the human surface protein CD59, were subjected to magnetic cell separation (MACS) in order to remove spontaneous mutants before irradiation with low-filtered 30 kVp (tungsten anode, 0.5mm Al filter) or conventional X-rays. Fractions of radiation-induced CD59 - mutants were quantified by flow-cytometry after immunofluorescence labelling of CD59 proteins. Results : Mammography X-rays were more effective than conventional X-rays at inducing killing of human fibroblasts, whereas 30 kVp X-rays and conventional X-rays were about equally effective at killing A L cells. Mutant frequencies were linearly related to dose in both mutation assays. An RBE = 2.7 was calculated for the yield of HPRT mutants in human fibroblasts exposed to mammography relative to conventional X-rays and an RBE = 2.4 was obtained for the CD59 mutant frequency in A L cells irradiated with low-filtered 30 kVp relative to conventional X-rays. Conclusions : Both low-filtered 30 kVp and mammography X-rays are mutagenic in mammalian cells in vitro. It is unknown if and how the enhanced mutagenicity of mammography X-rays measured in human cells in vitro translates into breast cancer risk for predisposed women with an enhanced inherited risk for breast cancer. Although the ICRP guidelines attribute the same relative biological effectiveness to all radiations of low LET, including X- and γ-radiations of all energies for radiobiological protection purposes including the assessment of risks in general terms, they also state that 'for the estimation of the likely consequences of an exposure of a known population, it will sometimes be better to use absorbed dose and specific data relating to the relative biological effectiveness of the radiations concerned and the probability coefficients relating to the exposed population' (ICRP 1991: §32). This latter statement may apply for the population of familial predisposed women. We hope that the presented data on the enhanced mutagenicity of mammography X-rays may stimulate a re-evaluation of the risk assessment of mammography for familial predisposed women. In the meantime, one should be cautious and avoid early and frequent mammography exposure of predisposed women. Alternative examination methods should be applied for these women with an inherited increased risk for breast cancer.

Purpose: The lung is the major dose-limiting organ for radiotherapy of cancer in the thoracic region. Immediate cellular damage after irradiation is supposed to result in cytokine-mediated multicellular interactions with induction and progression of inflammatory and fibrotic tissue reactions. Pentoxifylline (PTX) down-regulates the production of proinflammatory cytokines, particularly TNF-α, in response to noxious stimuli and may therefore provide protection against radiation-induced, cytokine-mediated cellular damage. The purpose of this study was to investigate the temporal and spatial release of TNF-α in the lung tissue after thoracic irradiation with 12Gy. In addition, we evaluated the ability of PTX to reduce the radiation-induced TNF-α release in this animal model of thoracic irradiation.Materials and methods: C57BL/6J mice were exposed to either sham irradiation or single fraction of 12Gy delivered to the thorax. Four study groups were defined: those that received neither irradiation nor PTX (NT group), those that received PTX but no irradiation (PTX group), those that underwent irradiation without PTX (XRT group) and those that received both PTX and irradiation (PTX/XRT group). Treated and sham-irradiated mice were sacrificed corresponding to the latent period and the pneumonic phase. The TNF-α mRNA expression in the lung tissue was quantified by ‘real-time’ quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Immunohistochemical detection methods (alkaline phosphatase anti-alkaline phosphatase (APAAP)) and automated image analysis were used for objective quantification of TNF-α protein expression.Results: Following thoracic irradiation with a single dose of 12Gy (XRT group), radiation-induced TNF-α mRNA release in the lung tissue was significantly increased during the acute phase of pneumonitis (P<0.05). The elevated levels of TNF-α mRNA during the pneumonic phase correlate with a significant increase of positive inflammatory cells, predominantly macrophages, in the lung parenchyma (P<0.05). In contrast to the radiation-only group (XRT-group), the lung tissue of the PTX-treated mice (PTX/XRT group) revealed only a minor radiation-mediated TNF-α response on mRNA and protein level.Conclusions: This study demonstrates a significant radiation-induced increase of TNF-α (on mRNA and protein level) in the lung tissue during the pneumonic phase. The predominant localisation of TNF-α in areas of inflammatory cell infiltrates suggests involvement of this cytokine in the pathogenesis of radiation-induced lung injury. In addition, we observed a pronounced reduction of the TNF-α mRNA and protein production in the study group that received both PTX and radiation (PTX/XRT group) as compared to the radiation-only group (XRT group). Therefore our results indicate that PTX down-regulates the TNF-α mRNA and protein production in the lung tissue in response to radiation.

Mutation induction in the HPRT gene of human fibroblasts after irradiation with mammography-like 29 kVp or 200 kVp x-rays shows radiohypersensitivity for doses smaller than approximately 0.5 Gy. Similarly, mutation induction in the CD 59 gene on human chromosome 11 in A(L) cells shows radiohypersensitivity for doses smaller than approximately 0.5 Gy after exposure to 200 kVp x-rays, but not after irradiation with low-filtered 30 kVp x-rays. The RBE values of 29 and 30 kVp x-rays relative to 200 kVp x-rays are strongly dose dependent. For neoplastic transformation of human hybrid (CGL1) cells after irradiation with 29 or 200 kVp x-rays or 60Co gamma rays a linear-quadratic dose relationship was observed with RBE values of approximately four and eight for mammography relative to 200 kVp x-rays and 60Co gamma rays, respectively.

Determination of the genotoxic effects of ionizing radiation, especially at low-doses, is of great importance for risk assessment, e.g. in radiological diagnostics. The human-hamster hybrid AL cell line has been shown previously to be a well-suited in vitro model for the study of mutations induced by various mutagens. The AL cells contain a standard set of hamster chromosomes and a single human chromosome 11, which confers the expression of the human cell surface protein CD59. Using CD59 specific antibodies, cells mutated in the CD59 gene can be detected and quantified by the loss of the cell surface marker. In contrast to previous studies, prior to irradiation we removed spontaneous mutants by magnetic cell separation (MACS) which allows analysis of radiation-induced mutation events only. We exposed AL cells to 100 kV X-rays at 0.1 to 5 Gy. The proportions of X-irradiation-induced CD59− mutants were quantified by flow cytometry after immunofluorescence labeling. Between 0.2 and 5 Gy the yield of CD59 mutants was a linear function of dose. The molecular analysis of individual CD59-negative clones induced after exposure of 1, 3 and 5 Gy of X-ray revealed a dose-dependent linear increase of large deletions (>6 Mbp), whereas, point mutations could be seen only in spontaneous CD59 mutants or after low-dose exposure (≤1 Gy). We conclude that the modified AL assay presented here is appropriate for detection and quantification of non-lethal DNA lesions induced by low-dose ionizing radiation.

Local control of the 77 transitional cell carcinoma of the bladder was analysed with respect to total dose after normalization for variations in fraction size and to overall treatment time. For the TCD50 of 63.3 Gy protraction of overall treatment time from 40 to 55 days average gave the decrease in local control rate from 50 % to about 5 %j The decrease in local tumour control with extension of overall treatment time likely reflects accelerated tumour repopulation during the treatment. To determine the time onset of the accelerated tumour clonogen repopulation, data taken from the literature were analysed. Results show that on average tumour clonogen repopulation in transitional cell cancer of the bladder accelerates after a lag period of about 5–6 weeks after the start of treatment and that a dose increment of 0.36 Gy per day is required to compensate for this repopulation. Such a dose increment is consistent with about 5–8 day clonogen doubling time. It suggests that overall treatment time is an important factor in the dose fractionation and protraction of time may have a significant impact on treatment outcome. Thus, radiotherapy for bladder cancer should be completed as soon as possible and total dose for high probability of local tumour control should consequently be delivered in more than 5 fractions per week.

PURPOSEThe lung is the major dose-limiting organ for radiotherapy of cancer in the thoracic region. The pathogenesis of radiation-induced lung injury at the molecular level is still unclear. Immediate cellular damage after irradiation is supposed to result in cytokine-mediated multicellular interactions with induction and progression of fibrotic tissue reactions. The purpose of this investigation was to evaluate the acute and long-term effects of radiation on the gene expression of transforming growth factor beta (TGF-beta) in a model of lung injury using fibrosis-sensitive C57BL/6 mice.METHODS AND MATERIALSThe thoraces of C57BL/6 mice were irradiated with 6 and 12 Gy, respectively. Treated and sham-irradiated control mice were sacrificed at times corresponding to the latent period (1, 3, 6, 12, 24, 48, 72 hours and 1 week postirradiation), the pneumonic phase (2, 4, 8, and 16 weeks postirradiation), and the beginning of the fibrotic phase (24 weeks postirradiation). The lung tissue from three different mice per dosage and time point was analyzed by a combination of polymerase chain reaction (PCR), immunohistochemistry, and light microscopy. The mRNA expression of TGF-beta was quantified by competitive reverse transcriptase/polymerase chain reaction (RT-PCR); the cellular origin of the TGF-beta protein was identified by immunohistochemical staining (alkaline phosphatase-anti-alkaline phosphatase [APAAP]). The cytokine expression on mRNA and protein level was correlated with the histopathological alterations.RESULTSFollowing thoracic irradiation with a single dose of 12 Gy, radiation-induced TGF-beta release in lung tissue was appreciable already within the first hours (1, 3, and 6 hours postirradiation) and reached a significant increase after 12 hours; subsequently (48 hours, 72 hours, and 1 week postirradiation) the TGF-beta expression declined to basal levels. At the beginning of the pneumonic phase, irradiation-mediated stimulation of TGF-beta release reached maximal values at 2 and 4 weeks. The elevated levels of TGF-beta mRNA during the latent phase have been found to correlate with immunohistochemical staining of alveolar macrophages. The most striking increase in TGF-beta immunoreactivity was seen during the acute phase of pneumonitis. Throughout this observation period, type II pneumocytes and fibroblasts (apart from inflammatory cells) served as important sources of TGF-beta expression. Increased TGF-beta expression was detected prominently in regions of histopathologic radiation injury. After exposure to a single radiation dose of 6 Gy, the lung tissue revealed only a minor radiation-mediated TGF-beta mRNA response. The modest upregulation ranged from 6 hours to 48 hours after irradiation. Corresponding to the only minor histopathologic changes after thoracic irradiation with 6 Gy, measurement of TGF-beta mRNA levels during the later time points revealed no significant alterations in comparison to untreated control mice.CONCLUSIONSThis study demonstrates an acute and long-lasting increase in the expression of TGF-beta in lung tissue following thoracic irradiation with 12 Gy. The predominant localization of TGF-beta in areas of inflammatory cell infiltrates and fibrosis suggests involvement of this cytokine in the pathogenesis of radiation-induced pulmonal fibrosis. Further studies should be performed to explore the role of other cytokines in the development of radiation injury. An improved understanding of the underlying mechanisms of pulmonary fibrosis may eventually lead to modulatory intervention at the molecular level to modify the fibrotic process.