Rspo1-LGR4 axis in BMSCs protects bone against radiation-induced injury through the mTOR-dependent autophagy pathway.

While mesenchymal stem cells (MSCs) have been widely used to repair radiation-induced bone damage, the molecular mechanism underlying the effects of MSCs in the maintenance of bone homeostasis under radiation stress remains largely unknown. In this study, the role and mechanisms of R-spondin 1 (Rspo1)-leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) axis on the initiation of self-defense of bone mesenchymal stem cells (BMSCs) and maintenance of bone homeostasis under radiation stress were investigated. Interestingly, radiation increased levels of Rspo1 and LGR4 in BMSCs. siRNA knockdown of Rspo1 or LGR4 aggravated radiation-induced impairment of self-renewal ability and osteogenic differentiation potential of BMSCs. However, exogenous Rspo1 significantly attenuated radiation-induced depletion of BMSCs, and promoted the lineage shift towards osteoblasts. This alteration was associated with the reversal of mammalian target of rapamycin (mTOR) activation and autophagy decrement. Pharmacological and genetic blockade of autophagy attenuated the radio-protective effects of Rspo1, rendering BMSCs more vulnerable to radiation-induced injury. Then bone radiation injury was induced in C57BL6J mice to further determine the radio-protective effects of Rspo1. In mice, administration of Rspo1 recombinant protein alleviated radiation-induced bone loss. Our results uncover that Rspo1-LGR4-mTOR-autophagy axis are key mechanisms by which BMSCs initiate self-defense against radiation and maintain bone homeostasis. Targeting Rspo1-LGR4 may provide a novel strategy for the intervention of radiation-induced bone damage.

NLRP3 protects mice from radiation-induced colon and skin damage via attenuating cGAS-STING signaling.

In the present study, the effects of NLRP3 on radiation-induced tissue damage, including colon and skin damage in mice, and the possible mechanisms were explored in vivo and in vitro. The mice were subjected to whole abdomen radiation by timed exposure to X-ray at a cumulative dose of 14 Gy. The survival rate showed that NLRP3 deficiency increased the mortality rate in mice. Furthermore, colon damage, evaluated by H&E staining and barrier function analysis, were significantly aggravated by NLRP3 deficiency. Enhanced phosphorylation of p-TBK1 and p-IRF3 in colonic tissue as well as elevated IFN-ß levels in the serum indicated hyperactivation of cGAS-STING signaling. Moreover, radiation-induced expression of p-TBK1, p-IRF3, and IFN-ß in BMDMs increased in vitro after NLRP3 knockout. Thus, our study outcomes suggest that NLRP3 may protect mice from radiation-induced tissue damage via attenuating cGAS-STING signaling.

Ionizing radiation induces myofibroblast differentiation via lactate dehydrogenase.

Pulmonary fibrosis is a common and dose-limiting side-effect of ionizing radiation used to treat cancers of the thoracic region. Few effective therapies are available for this disease. Pulmonary fibrosis is characterized by an accumulation of myofibroblasts and excess deposition of extracellular matrix proteins. Although prior studies have reported that ionizing radiation induces fibroblast to myofibroblast differentiation and collagen production, the mechanism remains unclear. Transforming growth factor-ß (TGF-ß) is a key profibrotic cytokine that drives myofibroblast differentiation and extracellular matrix production. However, its activation and precise role in radiation-induced fibrosis are poorly understood. Recently, we reported that lactate activates latent TGF-ß through a pH-dependent mechanism. Here, we wanted to test the hypothesis that ionizing radiation leads to excessive lactate production via expression of the enzyme lactate dehydrogenase-A (LDHA) to promote myofibroblast differentiation. We found that LDHA expression is increased in human and animal lung tissue exposed to ionizing radiation. We demonstrate that ionizing radiation induces LDHA, lactate production, and extracellular acidification in primary human lung fibroblasts in a dose-dependent manner. We also demonstrate that genetic and pharmacologic inhibition of LDHA protects against radiation-induced myofibroblast differentiation. Furthermore, LDHA inhibition protects from radiation-induced activation of TGF-ß. We propose a profibrotic feed forward loop, in which radiation induces LDHA expression and lactate production, which can lead to further activation of TGF-ß to drive the fibrotic process. These studies support the concept of LDHA as an important therapeutic target in radiation-induced pulmonary fibrosis.

The effect of RHIZOMA COPTIDIS and COPTIS CHINENSIS aqueous extract on radiation-induced skin injury in a rat model.

BACKGROUND: Radiation-induced skin injury is a common complication of radiotherapy. The RHIZOMA COPTIDIS and COPTIS CHINENSIS aqueous extract (RCE) can ameliorate radiation-induced skin injury in our clinical observation. But, the protective mechanism of RHIZOMA COPTIDIS and COPTIS CHINENSIS in radiation-induced skin injury remains unclear. METHODS: In this experiment, we developed a radiation-induced skin injury rat model to study the mechanism. The animals were randomly divided into control group, treatment group, radiation group, and treatment and radiation group. 5 rats in each group were separately executed on 2 d and 49 d post-radiation. The semi-quantitative skin injury score was used to measure skin reactions by unblinded observers, and hematoxylin and eosin staining was used to evaluate the damage areas by irradiation. The MDA content, SOD activity of skin and serum were measured to detect the oxidative stress. RESULTS: Acute skin reactions were caused by a single dose of 45 Gy of ß-ray irradiation, and the skin injury could be found in all rats receiving irradiation based on the observation of HE staining of skin at different time-points, while RCE could significantly ameliorate those changes. The MDA content in serum and skin of control rats was 4.13±0.12 mmol/ml and 4.95±0.35 mmol/mgprot on 2 d post-radiation. The rats receiving radiation showed an increased content of MDA (5.54±0.21 mmol/ml and 7.10±0.32 mmol/mgprot), while it was 4.57±0.21 mmol/ml and 5.95±0.24 mmol/mgprot after treated with RCE (p<0.05). Similar changes of the MDA content could be seen on 49 d post-radiation. However, the SOD activity of rats receiving radiation decreased compared with control group on both time-points, which was inhibited by RCE (p<0.05). Meanwhile, no valuable changes could be found between control group and treatment group on 2 d and 49 d. CONCLUSIONS: Our study provides evidences for the radioprotective role of RCE against radiation-induced skin damage in rats by modulating oxidative stress in skin, which may be a useful therapy for radiation-induced skin injury.

Mitigation of radiation induced pulmonary vascular injury by delayed treatment with captopril.

BACKGROUND AND OBJECTIVE: A single dose of 10 Gy radiation to the thorax of rats results in decreased total lung angiotensin-converting enzyme (ACE) activity, pulmonary artery distensibility and distal vascular density while increasing pulmonary vascular resistance (PVR) at 2 months post-exposure. In this study, we evaluate the potential of a renin-angiotensin system (RAS) modulator, the ACE inhibitor captopril, to mitigate this pulmonary vascular damage. METHODS: Rats exposed to 10 Gy thorax only irradiation and age-matched controls were studied 2 months after exposure, during the development of radiation pneumonitis. Rats were treated, either immediately or 2 weeks after radiation exposure, with two doses of the ACE inhibitor, captopril, dissolved in their drinking water. To determine pulmonary vascular responses, we measured pulmonary haemodynamics, lung ACE activity, pulmonary arterial distensibility and peripheral vessel density. RESULTS: Captopril, given at a vasoactive, but not a lower dose, mitigated radiation-induced pulmonary vascular injury. More importantly, these beneficial effects were observed even if drug therapy was delayed for up to 2 weeks after exposure. CONCLUSIONS: Captopril resulted in a reduction in pulmonary vascular injury that supports its use as a radiomitigator after an unexpected radiological event such as a nuclear accident.

Frozen human cells can record radiation damage accumulated during space flight: mutation induction and radioadaptation.

To estimate the space-radiation effects separately from other space-environmental effects such as microgravity, frozen human lymphoblastoid TK6 cells were sent to the "Kibo" module of the International Space Station (ISS), preserved under frozen condition during the mission and finally recovered to Earth (after a total of 134 days flight, 72 mSv). Biological assays were performed on the cells recovered to Earth. We observed a tendency of increase (2.3-fold) in thymidine kinase deficient (TK(-)) mutations over the ground control. Loss of heterozygosity (LOH) analysis on the mutants also demonstrated a tendency of increase in proportion of the large deletion (beyond the TK locus) events, 6/41 in the in-flight samples and 1/17 in the ground control. Furthermore, in-flight samples exhibited 48% of the ground-control level in TK(-) mutation frequency upon exposure to a subsequent 2 Gy dose of X-rays, suggesting a tendency of radioadaptation when compared with the ground-control samples. The tendency of radioadaptation was also supported by the post-flight assays on DNA double-strand break repair: a 1.8- and 1.7-fold higher efficiency of in-flight samples compared to ground control via non-homologous end-joining and homologous recombination, respectively. These observations suggest that this system can be used as a biodosimeter, because DNA damage generated by space radiation is considered to be accumulated in the cells preserved frozen during the mission, Furthermore, this system is also suggested to be applicable for evaluating various cellular responses to low-dose space radiation, providing a better understanding of biological space-radiation effects as well as estimation of health influences of future space explores.

p38 Mitogen-activated protein kinase signalling regulates vascular inflammation and epithelial barrier dysfunction in an experimental model of radiation-induced colitis.

BACKGROUND: : Microvascular injury and epithelial barrier dysfunction are rate-limiting aspects in radiation enteropathy. This study examined the role of p38 mitogen-activated protein kinase (p38 MAPK) signalling in radiation-induced colitis in an experimental model. METHODS: : The p38 MAPK inhibitor SB239063 was administered to mice immediately before exposure to 20 Gy radiation. Leucocyte- and platelet-endothelium interactions in the colonic microcirculation were assessed by intravital microscopy. Levels of myeloperoxidase (MPO) and CXC chemokines (macrophage inflammatory protein (MIP) 2 and cytokine-induced neutrophil chemoattractant (KC)), and albumin leakage were quantified 16 h after irradiation. RESULTS: : Irradiation induced an increase in leucocyte and platelet recruitment, MPO activity, CXC chemokine levels and intestinal leakage. Inhibition of p38 MAPK by SB239063 decreased radiation-induced leucocyte and platelet recruitment (leucocyte rolling and adhesion by 70 and 90 per cent, both P < 0.001; that of platelets by 70 and 74 per cent, both P < 0.001). It also reduced radiation-provoked increases in colonic MPO activity by 88 per cent (P < 0.001), formation of MIP-2 and KC by 72 and 74 per cent respectively (P = 0.003 and P < 0.001), and intestinal leakage by 81 per cent (P < 0.001). CONCLUSION: : p38 MAPK is an important signalling pathway in radiation-induced colitis.

[Protective activity of different concentration of tea polyphenols and its major compound EGCG against whole body irradiation-induced injury in mice].

OBJECTIVE: To evaluate the different concentrate of tea polyphenols (TP) and its compound for irradiation-protection and investigate its mechanism. METHOD: To evaluate the radioprotective activity, mice were exposed to whole body gamma irradiation. TP 80 and TP 50 (50, 10 mg x kg(-1)) and its major constituent epigallocatechin gallate (EGCG) (50, 10 mg x kg(-1)) were administered after irradiation to examine its inhibition against irradiation-induced injury. RESULT: This study indicate that in comparison with non-irradiated controls, irradiation resulted in a significant reduction the spleen index (spleen weight/body weight 100), haematological parameters (RBC, WBC and PLT), activity of superoxide dismutase (SOD), and increase of malondialdehde (MDA) level in 28 days. Oral administration of TP (50 mg x kg(-1)) shown the best effect on reducing the irradiation-induced injury on mice studied, and showed a protective effect against irradiation-induced haematological parameters (RBC, WBC and PLT), the spleen index and MDA level significant reduction, and antioxidase activity (SOD) decrease. CONCLUSION: The results suggest that TP 50 mg x kg(-1) and EGCG have in vivo antioxidant potential and radioprotective activity against whole body gamma irradiation in mice. It may be concluded that TP (50% EGCG) possess good irradiation-protective and antioxidant effect.

Role of phosphatidylinositol 3-kinase signaling pathway in radiation-induced liver injury.

Transforming growth factor-ß1 (TGF-ß1) is one of critical cytokines in radiation-induced liver injury. Hepatic stellate cells (HSC) are activated in the early stage of radiation-induced liver injury. However, it is currently unclear whether phosphatidylinositol 3-kinase (PI3K/Akt) signal pathway is activated in radiation-induced liver injury. Herein, male Sprague-Dawley rats were irradiated with 6 MV X-rays (30 Gy) on the right liver. Next, Hematoxylin and eosin staining, Masson staining, and electron microscopy were performed to examine pathological changes. Immunohistochemistry was performed to assess the expression of TGF-ß1, α-SMA, and p-Akt (S473) in liver tissues. In vitro, rat HSC cell line HSC-T6 cells were given different doses of 6 MV X-ray irradiation (10 and 20 Gy) and treated with LY294002. The expression of α-SMA and p-Akt in mRNA and protein levels were measured by reverse transcription-polymerase chain reactioin (RT-PCR) and Western blot. TGF-ß1 expression was detected by enzyme-linked immuno sorbent assay (ELISA). After irradiation, the liver tissues showed obvious pathological changes, indicating the establishment of the radiation-induced liver injury. Expression levels of TGF-ß1, α-SMA, and p-Akt (S473) protein in liver tissues were significantly increased after irradiation, and this increase was in a time-dependent manner, suggesting the activation of HSC and PI3K/Akt signal pathway. in vitro experiments showed that the TGF-ß1 secreted by HSCs, and the expression of Akt and α-SMA at mRNA and protein levels were significantly increased in irradiation groups. However, the expression of TGF-ß1, Akt, and α-SMA were significantly decreased in PI3K/Akt signal pathway inhibitor LY294002-treated group. Our results suggest that during radiation-induced liver injury, HSCs are activated by TGF-ß1-mediated PI3K/Akt signal pathway.

Granulocytes' enzymes as biomarkers of radiotoxicity in individuals occupationally exposed to low-level radiation.

PURPOSE: To investigate the possibility of using the granulocytes' enzymes alkaline phosphatase (L-ALP) and myeloperoxidase (MPO) as biomarkers to study and analyse contamination of nuclear medicine personnel working with radionuclides (RN) when radiotoxic effects are very low, before occupational radiation illness or benign haematological disorders and malignant diseases have occurred. Also, to investigate the relationship between chromosomal aberrations in lymphocytes and the activity of L-ALP and MPO in neutrophil granulocytes (NphG). MATERIALS AND METHODS: The absorbed external doses of ionizing radiation (IR) were measured by thermoluminescent personal dosimeters (TLD) for the duration of occupational exposure (DOE). Urine radioactivity was measured by gamma- spectrometry. Venous blood was used for leukocyte count and search for chromosomal aberrations by conventional cytogenetic techniques. Blood smears were stained for L-ALP and MPO using a modified Kaplow's method and the classical method with benzidine dihydrochloride, respectively. The occupationally exposed group (E) consisted of 74 workers exposed to short-life radioactive isotopes I131 (beta and mostly gamma emission) and mTc99 (gamma emission). The control group (C) consisted of 52 subjects living in the same region, working in the same institution, occupationally not exposed to RN. A patients' group (P; n=31) took I131 or mTc99 for diagnostic purposes. RESULTS: Although the measured values did not exceed the yearly quota for professionally exposed individuals, characteristic chromosomal aberrations in peripheral blood lymphocytes (dicentrics, fragments, rings) were identified. L-ALP and MPO activity was inhibited in the NphG in occupationally exposed workers, especially in persons with chromosomal aberrations, working for a long time in ionizing radiations zones (p <0.01). CONCLUSION: Decreased activity of L-ALP and MPO can reveal effects of long-lasting exposure to low-dose IR. A significant relationship between chromosomal aberrations in lymphocytes and activity of the enzymes in granulocytes was found.

Radiation-Induced Normal Tissue Damage: Oxidative Stress and Epigenetic Mechanisms.

Radiotherapy (RT) is currently one of the leading treatments for various cancers; however, it may cause damage to healthy tissue, with both short-term and long-term side effects. Severe radiation-induced normal tissue damage (RINTD) frequently has a significant influence on the progress of RT and the survival and prognosis of patients. The redox system has been shown to play an important role in the early and late effects of RINTD. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the main sources of RINTD. The free radicals produced by irradiation can upregulate several enzymes including nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase), lipoxygenases (LOXs), nitric oxide synthase (NOS), and cyclooxygenases (COXs). These enzymes are expressed in distinct ways in various cells, tissues, and organs and participate in the RINTD process through different regulatory mechanisms. In recent years, several studies have demonstrated that epigenetic modulators play an important role in the RINTD process. Epigenetic modifications primarily contain noncoding RNA regulation, histone modifications, and DNA methylation. In this article, we will review the role of oxidative stress and epigenetic mechanisms in radiation damage, and explore possible prophylactic and therapeutic strategies for RINTD.

Alteration of renal Na,K-ATPase in rats following the mediastinal γ-irradiation.

Na,K-ATPase represents the key enzyme that maintains the homeostasis of sodium and potassium ions in the cells. It was documented that in directly irradiated organs the activity of this enzyme is decreased. The aim of present study was to clarify the remote effect of irradiation in mediastinal area on the activity of the Na,K-ATPase in kidneys in rats. Ionizing radiation in single dose 25 Gy resulted in consequent decrease of the body weight gain as well as the size of kidneys in Wistar rats. In addition, radiation induced alterations in the oxidative status of blood plasma. Irradiation also decreased the activity of renal Na,K-ATPase. Measurements of enzyme kinetics that were dependent on the concentration of energy substrate ATP or cofactor Na+ indicated that the lowered enzyme activity is probably a consequence of decreased number of active molecules of the enzyme, as suggested by lowered Vmax values. Immunoblot analysis confirmed the lowered expression of the catalytic alpha subunit together with decreased content of the glycosylated form of beta subunit in the renal tissue of irradiated rats. The ability of the enzyme to bind the substrate ATP, as well as Na+ was not affected, as shown by unaltered values of Km and KNa . Irradiation of the body in the mediastinal area despite protection of kidneys by lead plates during application of X-ray was followed by significant decline of activity of the renal Na,K-ATPase, what may result in deteriorated homeostasis in the organism.

In-vivo optical imaging of hsp70 expression to assess collateral tissue damage associated with infrared laser ablation of skin.

Laser surgical ablation is achieved by selecting laser parameters that remove confined volumes of target tissue and cause minimal collateral damage. Previous studies have measured the effects of wavelength on ablation, but neglected to measure the cellular impact of ablation on cells outside the lethal zone. In this study, we use optical imaging in addition to conventional assessment techniques to evaluate lethal and sublethal collateral damage after ablative surgery with a free-electron laser (FEL). Heat shock protein (HSP) expression is used as a sensitive quantitative marker of sublethal damage in a transgenic mouse strain, with the hsp70 promoter driving luciferase and green fluorescent protein (GFP) expression (hsp70A1-L2G). To examine the wavelength dependence in the mid-IR, laser surgery is conducted on the hsp70A1-L2G mouse using wavelengths targeting water (OH stretch mode, 2.94 microm), protein (amide-II band, 6.45 microm), and both water and protein (amide-I band, 6.10 microm). For all wavelengths tested, the magnitude of hsp70 expression is dose-dependent and maximal 5 to 12 h after surgery. Tissues treated at 6.45 microm have approximately 4x higher hsp70 expression than 6.10 microm. Histology shows that under comparable fluences, tissue injury at the 2.94-microm wavelength was 2x and 3x deeper than 6.45 and 6.10 microm, respectively. The 6.10-microm wavelength generates the least amount of epidermal hyperplasia. Taken together, this data suggests that the 6.10-microm wavelength is a superior wavelength for laser ablation of skin.

Mitochondrial protein OPA mediates osteoporosis induced by radiation through the P38 signaling pathway.

OBJECTIVE: Bone marrow is full of mitochondria. However, the role of bone marrow mitochondrial protein in bone marrow damage and related signal transduction mechanism remains to be further studied. OPA is a newly discovered mitochondrial transmembrane protein. Its expression pattern and function in the physiological and pathological conditions of bone marrow are still elusive. The purpose of this study is to investigate the potential role of OPA in osteoporosis. PATIENTS AND METHODS: A mouse osteoporosis model was established by radiation. The OPA expression was tested by Western blot and qRT-PCR. The P38 signaling activity was evaluated by enzymatic activity kit. The mitochondrial ATP production was determined by flow cytometry. The bone marrow cell apoptosis was detected by flow cytometry. U0126 was used to pretreat mouse before modeling. Bone marrow tissue was collected from patients who received osteoporosis surgery to test the OPA expression, P38 activation and cell apoptosis. The OPA and P38 levels were analyzed by correlation. RESULTS: The mouse osteoporosis model was successfully established by radiation induction. In this osteoporosis model, the expression of OPA was increased. The P38 signaling was activated while the mitochondrial ATP production was reduced, with the increase of apoptosis of bone marrow cells. By contrast, U0126 pretreatment markedly inhibited the OPA expression, restrained the P38 signaling pathway, enhanced mitochondrial ATP production and suppressed the bone marrow cell apoptosis in mouse osteoporosis model. A significantly positive correlation was found between OPA and P38. CONCLUSIONS: The down-regulation of OPA inhibits cell apoptosis and improves osteoporosis via inducing mitochondrial ATP production and suppressing the P38 signaling pathway.

Radiotherapy-induced gut toxicity: Involvement of matrix metalloproteinases and the intestinal microvasculature.

Purpose To review the literature surrounding the involvement of the endothelium and matrix metalloproteinases (MMP) in radiotherapy-induced gut toxicity (RIGT) and further elucidate its complex pathobiology. Results RIGT involves damage to the gastrointestinal mucosa and is associated with diarrhoea, pain, and rectal bleeding depending on the area of exposure. The mechanisms underpinning RIGT are complex and have not yet been elucidated. Members of the MMP family, particularly MMP-2 and -9, have recently been identified as being key markers in RIGT and chemotherapy-induced gut toxicity (CIGT). Furthermore, the microvasculature has long been implicated in the development of toxicities following both chemotherapy and radiotherapy, however, the mechanisms behind this are yet to be explored. Conclusions It is proposed that matrix metalloproteinases are key regulators of endothelial mediators, and may play a key role in inducing damage to intestinal microvasculature following radiotherapy.

Effects of NOX1 on fibroblastic changes of endothelial cells in radiation­induced pulmonary fibrosis.

Lung fibrosis is a major complication in radiation­induced lung damage following thoracic radiotherapy, while the underlying mechanism has remained to be elucidated. The present study performed immunofluorescence and immunoblot assays on irradiated human pulmonary artery endothelial cells (HPAECs) with or without pre­treatment with VAS2870, a novel NADPH oxidase (NOX) inhibitor, or small hairpin (sh)RNA against NOX1, ­2 or ­4. VAS2870 reduced the cellular reactive oxygen species content induced by 5 Gy radiation in HPAECs and inhibited phenotypic changes in fibrotic cells, including increased alpha smooth muscle actin and vimentin, and decreased CD31 and vascular endothelial cadherin expression. These fibrotic changes were significantly inhibited by treatment with NOX1 shRNA, but not by NOX2 or NOX4 shRNA. Next, the role of NOX1 in pulmonary fibrosis development was assessed in the lung tissues of C57BL/6J mice following thoracic irradiation using trichrome staining. Administration of an NOX1­specific inhibitor suppressed radiation­induced collagen deposition and fibroblastic changes in the endothelial cells (ECs) of these mice. The results suggested that radiation­induced pulmonary fibrosis may be efficiently reduced by specific inhibition of NOX1, an effect mediated by reduction of fibrotic changes of ECs.

Elevated ARG1 expression in primary monocytes-derived macrophages as a predictor of radiation-induced acute skin toxicities in early breast cancer patients.

Radiation therapy (RT) the front-line treatment after surgery for early breast cancer patients is associated with acute skin toxicities in at least 40% of treated patients. Monocyte-derived macrophages are polarized into functionally distinct (M1 or M2) activated phenotypes at injury sites by specific systemic cytokines known to play a key role in the transition between damage and repair in irradiated tissues. The role of M1 and M2 macrophages in RT-induced acute skin toxicities remains to be defined. We investigated the potential value of M1 and M2 macrophages as predictive factors of RT-induced skin toxicities in early breast cancer patients treated with adjuvant RT after lumpectomy. Blood samples collected from patients enrolled in a prospective clinical study (n = 49) were analyzed at baseline and after the first delivered 2Gy RT dose. We designed an ex vivo culture system to differentiate patient blood monocytes into macrophages and treated them with M1 or M2-inducing cytokines before quantitative analysis of their "M1/M2" activation markers, iNOS, Arg1, and TGFß1. Statistical analysis was performed to correlate experimental data to clinical assessment of acute skin toxicity using Common Toxicity Criteria (CTC) grade for objective evaluation of skin reactions. Increased ARG1 mRNA significantly correlated with higher grades of erythema, moist desquamation, and CTC grade. Multivariate analysis revealed that increased ARG1 expression in macrophages after a single RT dose was an independent prognostic factor of erythema (p = 0 .032), moist desquamation (p = 0 .027), and CTC grade (p = 0 .056). Interestingly, multivariate analysis of ARG1 mRNA expression in macrophages stimulated with IL-4 also revealed independent prognostic value for predicting acute RT-induced toxicity factors, erythema (p = 0 .069), moist desquamation (p = 0 .037), and CTC grade (p = 0 .046). To conclude, our findings underline for the first time the biological significance of increased ARG1 mRNA levels as an early independent predictive biomarker of RT-induced acute skin toxicities.

Radiation-induced lung injury and inflammation in mice: role of inducible nitric oxide synthase and surfactant protein D.

Reactive nitrogen species (RNS) generated after exposure to radiation have been implicated in lung injury. Surfactant protein D (SP-D) is a pulmonary collectin that suppresses inducible nitric oxide synthase (iNOS)-mediated RNS production. Herein, we analyzed the role of iNOS and SP-D in radiation-induced lung injury. Exposure of wild-type (WT) mice to γ-radiation (8 Gy) caused acute lung injury and inflammation, as measured by increases in bronchoalveolar lavage (BAL) protein and cell content at 24 h. Radiation also caused alterations in SP-D structure at 24 h and 4 weeks post exposure. These responses were blunted in iNOS(-/-) mice. Conversely, loss of iNOS had no effect on radiation-induced expression of phospho-H2A.X or tumor necrosis factor (TNF)-α. Additionally, at 24 h post radiation, cyclooxygenase expression and BAL lipocalin-2 levels were increased in iNOS(-/-) mice, and heme oxygenase (HO)-1(+) and Ym1(+) macrophages were evident. Loss of SP-D resulted in increased numbers of enlarged HO-1(+) macrophages in the lung following radiation, along with upregulation of TNF-α, CCL2, and CXCL2, whereas expression of phospho-H2A.X was diminished. To determine if RNS play a role in the altered sensitivity of SP-D(-/-) mice to radiation, iNOS(-/-)/SP-D(-/-) mice were used. Radiation-induced injury, oxidative stress, and tissue repair were generally similar in iNOS(-/-)/SP-D(-/-) and SP-D(-/-) mice. In contrast, TNF-α, CCL2, and CXCL2 expression was attenuated. These data indicate that although iNOS is involved in radiation-induced injury and altered SP-D structure, in the absence of SP-D, it functions to promote proinflammatory signaling. Thus, multiple inflammatory pathways contribute to the pathogenic response to radiation.

Increased plasma levels of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in lung and breast cancer are altered during chest radiotherapy.

PURPOSE: Does the release of plasma matrix metalloproteinase-9 (MMP-9) by radiation-activated airway epithelial cells and infiltrating inflammatory cells play a role in the radiation damage or repair process in the lungs? We evaluated lung damage by ionizing radiation using plasma levels of MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and MMP-3 as biologic markers of tissue damage, and also their relationship to changes in pulmonary epithelial permeability, clinical signs and symptoms, and lung structural changes. METHODS AND MATERIALS: Seven serial studies were conducted in each of 8 patients undergoing chest radiotherapy (RT) for lung or breast cancer, beginning before the first treatment (baseline) and then biweekly to approximately 100 days during and after RT. Chest radiographs were monitored for each patient. Sandwich enzyme-linked immunoassays (ELISA) were used to measure plasma MMP-3, MMP-9, and TIMP-1 levels. Lung permeability was evaluated by measuring the rate of epithelial clearance of approximately 150 microCi ( approximately 5.6 MBq) inhaled (99m)Tc diethylenetriamine pentaacetate aerosol (DTPA). RESULTS: Lung and breast cancer resulted in very high plasma levels of MMP-9 (126-893 ng/mL) and TIMP-1 (496-8985 ng/mL) in all subjects studied before initiation of RT. This compares with plasma MMP-9 and TIMP-1 values in healthy volunteers of 29 +/- 11 ng/mL and 436 +/- 86 ng/mL, respectively. RT was followed by a sharp decrease in plasma MMP-9 within the first 2 weeks, but without a corresponding change in TIMP-1. In contrast, plasma MMP-3 levels, which are generally increased with inflammation, were elevated in only 1 of 5 subjects. CONCLUSION: Lung and breast cancer are associated with high plasma levels of MMP-9 and TIMP-1. These high baseline plasma levels of MMP-9 were reduced in the first 2 weeks of RT in 7 of 8 subjects, and TIMP-1 plasma levels remained high in all subjects. The decrease in plasma MMP-9 after initiation of chest RT appears to reflect a suppressive effect on cancer-induced cellular responses rather than a primary role for MMP-9 in radiation-induced lung damage. Likewise, the lack of a rise in plasma MMP-3 levels does not support a role for MMP-3 in tissue injury or repair in the lung. It remains to be determined whether plasma MMP-9 measurements will serve as a useful parameter in predicting cancer relapse.

Variation in the manganese superoxide dismutase gene (SOD2) is not a major cause of radiotherapy complications in breast cancer patients.

BACKGROUND AND PURPOSE: Small proportions of patients receiving radiotherapy develop marked long-term radiation damage. It is thought that this is due, at least in part, to intrinsic differences in cellular radiosensitivity, but the underlying mechanism is unknown. Reactive oxygen species are involved in cellular radiation damage, hence inter-individual differences in free radical detoxification may be related to radiosensitivity. Within mitochondria manganese superoxide dismutase (MnSOD) provides a major defence against oxidative damage by reactive oxygen species. MnSOD has been linked to expression of malignant phenotype and apoptosis and polymorphic variation in the gene, SOD2 to risk of breast cancer. MATERIALS AND METHODS: Forty-one breast cancer patients developing marked changes in breast appearance after radiotherapy and 39 patients who showed no clinically detectable reaction after radiotherapy were analyzed for germline sequence variation in SOD2. RESULTS: The Ala-9Val polymorphism was detected, but no other sequence variants were detected in SOD2. Both alleles of the Ala-9Val polymorphism were equally distributed between the two patient groups. CONCLUSIONS: Sequence variation in SOD2 is not the major cause of radiotherapy complications in women with breast cancer.