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1.  Effects of radiation on the epidermal growth factor receptor pathway in the heart 
Purpose
Radiation-induced heart disease (RIHD) is a serious side effect of thoracic radiotherapy. The epidermal growth factor receptor (EGFR) pathway is essential for the function and survival of cardiomyocytes. Hence, agents that target the EGFR pathway are cardiotoxic. Tocotrienols protect from radiation injury, but may also enhance the therapeutic effects of EGFR pathway inhibitors in cancer treatment. This study investigates the effects of local irradiation on the EGFR pathway in the heart and tests whether tocotrienols may modify radiation-induced changes in this pathway.
Methods
Male Sprague-Dawley rats received image-guided localized heart irradiation with 21 Gy. Twenty four hours before irradiation, rats received a single dose of tocotrienol-enriched formulation or vehicle by oral gavage. At time points from 2 hours to 9 months after irradiation, left ventricular expression of EGFR pathway mediators was studied.
Results
Irradiation caused a decrease in the expression of epidermal growth factor (EGF) and neuregulin-1 (Nrg-1) mRNA from 6 hours up to 10 weeks, followed by an upregulation of these ligands and the receptor erythroblastic leukemia viral oncogene homolog (ErbB)4 at 6 months. In addition, the upregulation of Nrg-1 was statistically significant up to 9 months after irradiation. A long-term upregulation of ErbB2 protein did not coincide with changes in transcription or post-translational interaction with the chaperone heat shock protein 90 (HSP90). Pretreatment with tocotrienols prevented radiation-induced changes at 2 weeks.
Conclusions
Local heart irradiation causes long-term changes in the EGFR pathway. Studies have to address how radiation may interact with cardiotoxic effects of EGFR inhibitors.
doi:10.3109/09553002.2013.782110
PMCID: PMC3700655  PMID: 23488537
Radiation-induced heart disease; Epidermal Growth Factor Receptor pathway; Neuregulin-1; Tocotrienols
2.  Combined radiation and burn injury results in exaggerated early pulmonary inflammation 
Radiation research  2013;180(3):276-283.
Events such as a nuclear meltdown accident or nuclear attack have potential for severe radiation injuries. Radiation injury frequently occurs in combination with other forms of trauma, most often burns. Thus far, combined injury studies have focused mainly on skin wound healing and damage to the gut. Since both radiation exposure and remote burn have pulmonary consequences, we examined the early effects of combined injury on the lung. C57BL/6 male mice were subjected to 5 Gy of total body irradiation followed by a 15% total body surface area scald burn. Lungs from surviving animals were examined for evidence of inflammation and pneumonitis. At 48 hours post-injury, pathology of the lungs from combined injury mice showed greater inflammation compared to all other treatment groups, with marked red blood cell and leukocyte congestion of the pulmonary vasculature. There was excessive leukocyte accumulation, primarily neutrophils, in the vasculature and interstitium, with occasional cells in the alveolar space. At 24 and 48 hours post-injury, myeloperoxidase levels in lungs of mice given combined injury were elevated compared to all other treatment groups (p<0.01), confirming histological evidence of neutrophil accumulation. Pulmonary levels of the neutrophil chemoattractant KC (CXCL1) were 3 times above that of either injury alone (p<0.05). Further, monocyte chemotactic protein-1 (MCP-1, CCL2) was increased 2-fold and 3-fold compared to burn injury or radiation injury, respectively (p<0.05). Together, these data suggest that combined radiation and burn injury augments early pulmonary congestion and inflammation.. Currently, countermeasures for this unique type of injury are extremely limited. Further research is needed to elucidate the mechanisms behind the synergistic effects of combined injury in order to develop appropriate treatments.
doi:10.1667/RR3104.1
PMCID: PMC4011563  PMID: 23899376
radiation injury; burn injury; severe combined injury; cytokines; pneumonitis
3.  C/EBPδ Deficiency Sensitizes Mice to Ionizing Radiation-Induced Hematopoietic and Intestinal Injury 
PLoS ONE  2014;9(4):e94967.
Knowledge of the mechanisms involved in the radiation response is critical for developing interventions to mitigate radiation-induced injury to normal tissues. Exposure to radiation leads to increased oxidative stress, DNA-damage, genomic instability and inflammation. The transcription factor CCAAT/enhancer binding protein delta (Cebpd; C/EBPδ is implicated in regulation of these same processes, but its role in radiation response is not known. We investigated the role of C/EBPδ in radiation-induced hematopoietic and intestinal injury using a Cebpd knockout mouse model. Cebpd−/− mice showed increased lethality at 7.4 and 8.5 Gy total-body irradiation (TBI), compared to Cebpd+/+ mice. Two weeks after a 6 Gy dose of TBI, Cebpd−/− mice showed decreased recovery of white blood cells, neutrophils, platelets, myeloid cells and bone marrow mononuclear cells, decreased colony-forming ability of bone marrow progenitor cells, and increased apoptosis of hematopoietic progenitor and stem cells compared to Cebpd+/+ controls. Cebpd−/− mice exhibited a significant dose-dependent decrease in intestinal crypt survival and in plasma citrulline levels compared to Cebpd+/+ mice after exposure to radiation. This was accompanied by significantly decreased expression of γ-H2AX in Cebpd−/− intestinal crypts and villi at 1 h post-TBI, increased mitotic index at 24 h post-TBI, and increase in apoptosis in intestinal crypts and stromal cells of Cebpd−/− compared to Cebpd+/+ mice at 4 h post-irradiation. This study uncovers a novel biological function for C/EBPδ in promoting the response to radiation-induced DNA-damage and in protecting hematopoietic and intestinal tissues from radiation-induced injury.
doi:10.1371/journal.pone.0094967
PMCID: PMC3991713  PMID: 24747529
4.  Metabolomic Changes in Gastrointestinal Tissues after Whole Body Radiation in a Murine Model 
Molecular bioSystems  2013;9(4):723-731.
Exposure to ionizing radiation (IR) elicits a set of complex biological responses involving gene expression and protein turnover that ultimately manifest as dysregulation of metabolic processes representing the cellular phenotype. Although radiation biomarkers have been reported in urine and serum, they are not informative about IR mediated tissue or organ specific injury. In the present study we report IR induced metabolic changes in gastrointestinal (GI) tissue of CD2F1 mice using ultra-performance liquid chromatography (UPLC) coupled with electrospray time-of-flight mass spectrometry. Post-radiation GI injury is a critical determinant of survival after exposure to IR. Our results show a distinct dose and time dependent response to GI tissue injury.
doi:10.1039/c3mb25454b
PMCID: PMC3601576  PMID: 23403731
5.  BMS-345541 Sensitizes MCF-7 Breast Cancer Cells to Ionizing Radiation by Selective Inhibition of Homologous Recombinational Repair of DNA Double-Strand Breaks 
Radiation research  2012;179(2):160-170.
Our study was to elucidate the mechanisms whereby BMS-345541 (BMS, a specific IκB kinase β inhibitor) inhibits the repair of DNA double-strand breaks (DSBs) and evaluate whether BMS can sensitize MCF-7 breast cancer cells (MCF-7 cells) to ionizing radiation (IR) in an apoptosis-independent manner. In this study, MCF-7 cells were exposed to IR in vitro and in vivo with or without pretreatment of BMS. The effects of BMS on the repair of IR-induced DSBs by homologous recombination (HR) and non-homologous end-joining (NHEJ) were analyzed by the DR-GFP and EJ5-GFP reporter assays and IR-induced γ-H2AX, 53BP1, Brca1 and Rad51 foci assays. The mechanisms by which BMS inhibits HR were examined by microarray analysis and quantitative reverse transcription PCR. The effects of BMS on the sensitivity of MCF-7 cells to IR were determined by MTT and clonogenic assays in vitro and tumor growth inhibition in vivo in a xenograft mouse model. The results showed that BMS selectively inhibited HR repair of DSBs in MCF-7 cells, most likely by down-regulation of several genes that participate in HR. This resulted in a significant increase in the DNA damage response that sensitizes MCF-7 cells to IR-induced cell death in an apoptosis-independent manner. Furthermore, BMS treatment sensitized MCF-7 xenograft tumors to radiation therapy in vivo in an association with a significant delay in the repair of IR-induced DSBs. These data suggest that BMS is a novel HR inhibitor that has the potential to be used as a radiosensitizer to increase the responsiveness of cancer to radiotherapy.
doi:10.1667/RR3034.1
PMCID: PMC3607209  PMID: 23259762
6.  Inhibition of protease-activated receptor 1 ameliorates intestinal radiation mucositis in a preclinical rat model 
SUMMARY
Direct inhibition of thrombin has a beneficial effect on intestinal radiation toxicity. A small molecule inhibitor of protease-activated receptor 1 (PAR1), the most relevant receptor for thrombin, was administered to rats undergoing localized, fractionated irradiation of a segment of small intestine. Exogenous administration of the PAR1 inhibitor substantially ameliorated acute intestinal radiation mucositis, but did not interrupt the subsequent development of intestinal radiation fibrosis.
Purpose
Inhibition of thrombin ameliorates intestinal radiation injury. Protease-activated receptor 1 (PAR1), is the most relevant receptor for thrombin signaling and is not expressed on rat platelets. We used a specific small molecule inhibitor of PAR1 signaling to determine whether the beneficial effect of thrombin inhibition on radiation enteropathy development is due to inhibition of blood clotting or to cellular (PAR1-mediated) thrombin effects.
Methods and Materials
Rats underwent fractionated X-irradiation (5 Gy x 9) of a 4-cm small bowel segment. Early radiation toxicity was evaluated in rats receiving PAR1 inhibitor (SCH602539, 0, 10, or 15 μg/kg/d) from 1 day before to 2 weeks after the end of irradiation. The effect of PAR1 inhibition on development of chronic intestinal radiation fibrosis was evaluated in animals receiving SCH602539 (0, 15, or 30 μg/kg/d) until 2 weeks after irradiation, or continuously until termination of the experiment 26 weeks after irradiation.
Results
PAR1 blockade ameliorated early intestinal toxicity, with reduced overall intestinal radiation injury (p=0.002), number of myeloperoxidase-positive (p=0.03) and proliferating cell nuclear antigen-positive (p=0.04) cells, and collagen III accumulation (p=0.005). In contrast, there was no difference in delayed radiation enteropathy in either the 2- or 26-week administration groups.
Conclusion
Pharmacological blockade of PAR1 appears to reduce early radiation mucositis, but does not affect the level of delayed intestinal radiation fibrosis. Early radiation enteropathy is related to activation of cellular thrombin receptors, whereas, platelet activation or fibrin formation may play a greater role for the development of delayed toxicity. Because of the favorable side effect profile, PAR1 blockade should be further explored as a method to ameliorate acute intestinal radiation toxicity in patients undergoing radiation therapy for cancer and to protect first responders and rescue personnel in radiological/nuclear emergencies.
doi:10.1016/j.ijrobp.2012.02.007
PMCID: PMC3462888  PMID: 22580123
Intestines; radiation injuries; thrombin; thrombin receptors; protease-activated receptors
7.  Glutathione Preservation during Storage of Rat Lenses in Optisol-GS and Castor Oil 
PLoS ONE  2013;8(11):e79620.
Background
Glutathione concentration in the lens decreases in aging and cataractous lenses, providing a marker for tissue condition. Experimental procedures requiring unfrozen lenses from donor banks rely on transportation in storage medium, affecting lens homeostasis and alterations in glutathione levels. The aim of the study was to examine the effects of Optisol-GS and castor oil on lens condition, determined from their ability to maintain glutathione concentrations.
Methodology/Principal Findings
Rat lenses were stored in the two types of storage media at varying time intervals up to 3 days. Glutathione concentration was afterwards determined in an enzymatic detection assay, specific for both reduced and oxidized forms. Lenses removed immediately after death exhibited a glutathione concentration of 4.70±0.29 mM. In vitro stored lenses in Optisol-GS lost glutathione quickly, ending with a concentration of 0.60±0.34 mM after 3 days while castor oil stored lenses exhibited a slower decline and ended at 3 times the concentration. A group of lenses were additionally stored under post mortem conditions within the host for 6 hours before its removal. Total glutathione after 6 hours was similar to that of lenses removed immediately after death, but with altered GSH and GSSG concentrations. Subsequent storage of these lenses in media showed changes similar to those in the first series of experiments, albeit to a lesser degree.
Conclusions/Significance
It was determined that storage in Optisol-GS resulted in a higher loss of glutathione than lenses stored in castor oil. Storage for more than 12 hours reduced glutathione to half its original concentration, and was considered unusable after 24 hours.
doi:10.1371/journal.pone.0079620
PMCID: PMC3834120  PMID: 24260265
8.  Enhanced implementation of low back pain guidelines in general practice: study protocol of a cluster randomised controlled trial 
Background
Evidence-based clinical practice guidelines may improve treatment quality, but the uptake of guideline recommendations is often incomplete and slow. Recently new low back pain guidelines are being launched in Denmark. The guidelines are considered to reduce personal and public costs. The aim of this study is to evaluate whether a complex, multifaceted implementation strategy of the low back pain guidelines will reduce secondary care referral and improve patient outcomes compared to the usual simple implementation strategy.
Methods/design
In a two-armed cluster randomised trial, 100 general practices (clusters) and 2,700 patients aged 18 to 65 years from the North Denmark region will be included. Practices are randomly allocated 1:1 to a simple or a complex implementation strategy. Intervention practices will receive a complex implementation strategy, including guideline facilitator visits, stratification tools, and quality reports on low back pain treatment. Primary outcome is referral to secondary care. Secondary outcomes are pain, physical function, health-related quality of life, patient satisfaction with care and treatment outcome, employment status, and sick leave. Primary and secondary outcomes pertain to the patient level. Assessments of outcomes are blinded and follow the intention-to-treat principle. Additionally, a process assessment will evaluate the degree to which the intervention elements will be delivered as planned, as well as measure changes in beliefs and behaviours among general practitioners and patients.
Discussion
This study provides knowledge concerning the process and effect of an intervention to implement low back pain guidelines in general practice, and will provide insight on essential elements to include in future implementation strategies in general practice.
Trial registration
Registered as NCT01699256 on ClinicalTrials.gov.
doi:10.1186/1748-5908-8-124
PMCID: PMC4015716  PMID: 24139140
General practice; Intervention studies; Guideline; Health plan implementation; Low back pain; Referral; Consultation
9.  Cardiac inflammation after local irradiation is influenced by the kallikrein-kinin system 
Cancer research  2012;72(19):4984-4992.
Radiotherapy of intrathoracic and chest wall tumors may lead to exposure of the heart to ionizing radiation, resulting in radiation-induced heart diseases (RIHD). The main manifestations of RIHD become apparent many years after treatment and include cardiomyopathy and accelerated atherosclerosis. This study examines the role of the kallikrein-kinin system (KKS) in RIHD by investigating the cardiac radiation response in a kininogen-deficient Brown Norway Katholiek (BN/Ka) rat model. BN/Ka rats and wild-type Brown Norway (BN) rats were exposed to local heart irradiation with a single dose of 18 Gy or 24 Gy and were observed for 3-6 months. Examinations included in vivo and ex vivo cardiac function, histopathology, gene and protein expression measurements, and mitochondrial swelling assays. Upon local heart irradiation, changes in in vivo cardiac function were significantly less in BN/Ka rats. For instance, a single dose of 24 Gy caused a 35% increase in fractional shortening in BN rats compared to a 16% increase in BN/Ka rats. BN rats, but not BN/Ka rats, showed a 56% reduction in cardiac numbers of CD2-positive cells, and a 57% increase in CD68-positive cells, together with a 52% increase in phosphorylation of Erk1/2. Local heart irradiation had similar effects on histopathology, mitochondrial changes, and left ventricular mRNA levels of NADPH oxidases in the two genotypes. These results suggest that the KKS plays a role in the effects of radiation on cardiac function and recruitment of inflammatory cells. The KKS may have these effects at least in part by altering Erk1/2 signaling.
doi:10.1158/0008-5472.CAN-12-1831
PMCID: PMC3463770  PMID: 22865451
Radiation-induced heart disease; Kallikrein-kinin system; Bradykinin
10.  Procalcitonin as a predictive biomarker for total body irradiation induced bacterial load and lethality in mice 
Shock (Augusta, Ga.)  2012;38(2):170-176.
Sepsis is the leading cause of mortality in intensive care units. Early detection and intervention are critical to prevent death. The acute radiation syndrome is characterized by damage of the gastrointestinal and hematopoietic systems. Translocation of intestinal microflora combined with immune system compromise may lead to septicemia and death. This work examined the utility of procalcitonin, a clinical sepsis biomarker, in a mouse model of radiation toxicity.
C57/BL6 mice were exposed to total body irradiation (TBI). Intestinal mucosal permeability was measured in vivo, and liver bacterial load and plasma levels of procalcitonin (PCT), lipopolysaccharide (LPS), and lipopolysaccharide binding protein (LBP) were measured at baseline and 3.5, 7, and 10 days after TBI. The value of early PCT in predicting subsequent lethality was determined by receiver operator characteristics (ROC) analysis.
Four days after TBI a dose-dependent increase in permeability of the intestinal mucosa was observed, while bacterial translocation was present from day 7 onward. There was a high positive correlation between bacterial translocation and all sepsis biomarkers, with PCT exhibiting the strongest correlation. Moreover, plasma PCT levels were elevated already from day 3.5 onwards, whereas, LPS was elevated from day 7 and LBP only 10 days after TBI. ROC analysis revealed that PCT levels measured 3.5 days after TBI predicted lethality at 10 days.
These data demonstrate the value of PCT as an early biomarker in radiation-induced bacteremia for mouse studies and suggest that clinical results from other septic conditions may apply to post-radiation septicemia in humans.
doi:10.1097/SHK.0b013e31825b2db3
PMCID: PMC3399035  PMID: 22576002
radiation; acute radiation syndromes; procalcitonin; biomarker; endotoxin; bacteremia; lipopolysaccharide
11.  Pharmacological Induction of Transforming Growth Factor-Beta1 in Rat Models Enhances Radiation Injury in the Intestine and the Heart 
PLoS ONE  2013;8(7):e70479.
Radiation therapy in the treatment of cancer is dose limited by radiation injury in normal tissues such as the intestine and the heart. To identify the mechanistic involvement of transforming growth factor-beta 1 (TGF-β1) in intestinal and cardiac radiation injury, we studied the influence of pharmacological induction of TGF-β1 with xaliproden (SR 57746A) in rat models of radiation enteropathy and radiation-induced heart disease (RIHD). Because it was uncertain to what extent TGF-β induction may enhance radiation injury in heart and intestine, animals were exposed to irradiation schedules that cause mild to moderate (acute) radiation injury. In the radiation enteropathy model, male Sprague-Dawley rats received local irradiation of a 4-cm loop of rat ileum with 7 once-daily fractions of 5.6 Gy, and intestinal injury was assessed at 2 weeks and 12 weeks after irradiation. In the RIHD model, male Sprague-Dawley rats received local heart irradiation with a single dose of 18 Gy and were followed for 6 months after irradiation. Rats were treated orally with xaliproden starting 3 days before irradiation until the end of the experiments. Treatment with xaliproden increased circulating TGF-β1 levels by 300% and significantly induced expression of TGF-β1 and TGF-β1 target genes in the irradiated intestine and heart. Various radiation-induced structural changes in the intestine at 2 and 12 weeks were significantly enhanced with TGF-β1 induction. Similarly, in the RIHD model induction of TGF-β1 augmented radiation-induced changes in cardiac function and myocardial fibrosis. These results lend further support for the direct involvement of TGF-β1 in biological mechanisms of radiation-induced adverse remodeling in the intestine and the heart.
doi:10.1371/journal.pone.0070479
PMCID: PMC3723823  PMID: 23936211
12.  Effects of Late Administration of Pentoxifylline and Tocotrienols in an Image-Guided Rat Model of Localized Heart Irradiation 
PLoS ONE  2013;8(7):e68762.
Radiation-induced heart disease (RIHD) is a long-term side effect of radiotherapy of intrathoracic, chest wall and breast tumors when radiation fields encompass all or part of the heart. Previous studies have shown that pentoxifylline (PTX) in combination with α-tocopherol reduced manifestations of RIHD in rat models of local heart irradiation. The relative contribution of PTX and α-tocopherol to these beneficial effects are not known. This study examined the effects of PTX alone or in combination with tocotrienols, forms of vitamin E with potential potent radiation mitigation properties. Rats received localized X-irradiation of the heart with an image-guided irradiation technique. At 3 months after irradiation rats received oral treatment with vehicle, PTX, or PTX in combination with a tocotrienol-enriched formulation. At 6 months after irradiation, PTX-treated rats showed arrhythmia in 5 out of 14 animals. PTX alone or in combination with tocotrienols did not alter cardiac radiation fibrosis, left ventricular protein expression of the endothelial markers von Willebrand factor and neuregulin-1, or phosphorylation of the signal mediators Akt, Erk1/2, or PKCα. On the other hand, tocotrienols reduced cardiac numbers of mast cells and macrophages, but enhanced the expression of tissue factor. While this new rat model of localized heart irradiation does not support the use of PTX alone, the effects of tocotrienols on chronic manifestations of RIHD deserve further investigation.
doi:10.1371/journal.pone.0068762
PMCID: PMC3718790  PMID: 23894340
13.  Synergistic Radioprotection by Gamma-Tocotrienol and Pentoxifylline: Role of cAMP Signaling 
ISRN Radiology  2013;2013:390379.
Purpose. This study was designed to determine the efficacy and mechanisms of radioprotection by the combination of gamma-tocotrienol (GT3) and pentoxifylline (PTX) against acute radiation injury. Materials and Methods. Post-irradiation survival was monitored to determine the most efficacious dose and time of administration of PTX. Dose reduction factor (DRF) was calculated to compare the radioprotective efficacy of the combination. To determine the mechanism of synergistic radioprotection by the combination, mevalonate or calmodulin were coadministered with the GT3-PTX combination. Mevalonate was used to reverse the inhibitory effect of GT3 on 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), and calmodulin was used to reverse the inhibition of phosphodiesterase (PDE) by PTX. Results. The combination was most effective when 200 mg/kg of PTX was administered 15 min before irradiation along with 200 mg/kg of GT3 (−24 h) and resulted in a DRF of 1.5. White blood cells and neutrophil counts showed accelerated recovery in GT3-PTX-treated groups compared to GT3. Mevalonate had no effect on the radioprotection of GT3-PTX; calmodulin abrogated the synergistic radioprotection by GT3-PTX. Conclusion. The mechanism of radioprotection by GT3-PTX may involve PDE inhibition.
doi:10.5402/2013/390379
PMCID: PMC4045513  PMID: 24959559
14.  Novel drugs to ameliorate gastro-intestinal normal tissue radiation toxicity in clinical practice: what is emerging from the laboratory 
Purpose of the review
To give an overview of promising novel agents under development for the prevention and reduction of gastro-intestinal radiation injury.
Recent findings
Currently, several novel agents are being tested as drugs to prevent or reduce gastro-intestinal radiation injury. These drugs may not only prevent injury, but may also mitigate toxicity, i.e. reduce injury after radiation exposure has occurred. Promising novel agents include the somatostatin analogue SOM230, growth factors, agents acting on the toll-like receptor 5 pathway, endothelial protectants, and the vitamin E analogue γ-tocotrienol.
Summary
Gastro-intestinal radiation injury is the most important dose limiting factor during radiotherapy of the abdomen or pelvis. It may severely affect quality of life both during radiotherapy treatment and in cancer survivors. To date, there are no agents that can prevent or reduce intestinal radiation injury. Hence, there is an urgent need for the development of novel drugs to ameliorate intestinal toxicity during and after radiotherapy. This review summarizes several agents that have been shown to reduce intestinal radiation injury in animals. Further research is needed to investigate their safety and efficacy in patients receiving radiotherapy for abdominal or pelvic tumours.
doi:10.1097/SPC.0b013e32834e3bd7
PMCID: PMC3677768  PMID: 22228028
radiation injuries; radioprotection; somatostatin; growth factors; toll-like receptor; γ-tocotrienol
15.  Statistical Analysis of Survival Data From Radiation Countermeasure Experiments 
Radiation Research  2012;177(5):546-554.
We present an introduction to, and examples of, Cox proportional hazards regression in the context of animal lethality studies of potential radioprotective agents. This established method is seldom used to analyze survival data collected in such studies, but is appropriate in many instances. Presenting a hypothetical radiation study that examines the efficacy of a potential radioprotectant both in the absence and presence of a potential modifier, we detail how to implement and interpret results from a Cox proportional hazards regression analysis used to analyze the survival data, and we provide relevant SAS® code. Cox proportional hazards regression analysis of survival data from lethal radiation experiments (1) considers the whole distribution of survival times rather than simply the commonly used proportions of animals that survived, (2) provides a unified analysis when multiple factors are present, and (3) can increase statistical power by combining information across different levels of a factor. Cox proportional hazards regression should be considered as a potential statistical method in the toolbox of radiation researchers.
PMCID: PMC3387733  PMID: 22401302
16.  Laser Capture Microdissected Mucosa versus Whole Tissue Specimens for Assessment of Radiation-Induced Dynamic Molecular and Pathway Changes in the Small Intestine 
PLoS ONE  2013;8(1):e53711.
Background
The intestinal mucosa is the compartment that sustains the most severe injury in response to radiation and is therefore of primary interest. The use of whole gut extracts for analysis of gene expression may confound important changes in the mucosa. On the other hand, laser capture microdissection (LCM) is hampered by the unstable nature of RNA and by a more complicated collection process. This study assessed, in parallel samples from a validated radiation model, the indications for use of LCM for intestinal gene expression analysis.
Methodology/Principal Findings
RNA was extracted from mouse whole intestine and from mucosa by LCM at baseline and 4 h, 24 h, and 3.5 d after total body irradiation and subjected to microarray analysis. Among mucosal genes that were altered > = 2-fold, less than 7% were present in the whole gut at 4 and 24 h, and 25% at 3.5 d. As expected, pathway analysis of mucosal LCM samples showed that radiation activated the coagulation system, lymphocyte apoptosis, and tight junction signaling, and caused extensive up-regulation of cell cycle and DNA damage repair pathways. Using similar stringent criteria, regulation of these pathways, with exception of the p53 pathway, was undetectable in the whole gut. Radiation induced a dramatic increase of caspase14 and ectodysplasin A2 receptor (Eda2r), a TNFα receptor, in both types of samples.
Conclusions/Significance
LCM-isolated mucosal specimens should be used to study cellular injury, cell cycle control, and DNA damage repair pathways. The remarkable increase of caspase14 and Eda2r suggests a novel role for these genes in regulating intestinal radiation injury. Comparative gene expression data from complex tissues should be interpreted with caution.
doi:10.1371/journal.pone.0053711
PMCID: PMC3544848  PMID: 23341980
18.  The helicase and ATPase activities of RECQL4 are compromised by mutations reported in three human patients 
Aging (Albany NY)  2012;4(11):790-802.
RECQL4 is one of five members of the human RecQ helicase family, and is implicated in three syndromes displaying accelerating aging, developmental abnormalities and a predisposition to cancer. In this study, we purified three variants of RECQL4 carrying previously reported patient mutations. These three mutant proteins were analyzed for the known biochemical activities of RECQL4: DNA binding, unwinding of duplex DNA, ATP hydrolysis and annealing of simplex DNA. Further, the mutant proteins were evaluated for stability and recruitment to sites of laser-induced DNA damage. One mutant was helicase-dead, had marginal ATPase activity and may be structurally compromised, while the other two showed greatly reduced helicase and ATPase activities. The remaining biochemical activities and ability to recruit to damage sites were not significantly impaired for any of the mutants. Our findings demonstrate a consistent pattern of functional deficiency and provide further support for a helicase-dependent cellular function of RECQL4 in addition to its Nterminus-dependent role in initiation of replication, a function that may underlie the phenotype of RECQL4-linked disease.
PMCID: PMC3560432  PMID: 23238538
RecQ helicase; RECQL4; Rothmund-Thomson Syndrome; RAPADILINO Syndrome
19.  Vitamin D Metabolism and Effects on Pluripotency Genes and Cell Differentiation in Testicular Germ Cell Tumors In Vitro and In Vivo12 
Neoplasia (New York, N.Y.)  2012;14(10):952-963.
Testicular germ cell tumors (TGCTs) are classified as either seminomas or nonseminomas. Both tumors originate from carcinoma in situ (CIS) cells, which are derived from transformed fetal gonocytes. CIS, seminoma, and the undifferentiated embryonal carcinoma (EC) retain an embryonic phenotype and express pluripotency factors (NANOG/OCT4). Vitamin D (VD) is metabolized in the testes, and here, we examined VD metabolism in TGCT differentiation and pluripotency regulation. We established that the VD receptor (VDR) and VD-metabolizing enzymes are expressed in human fetal germ cells, CIS, and invasive TGCTs. VD metabolism diminished markedly during the malignant transformation from CIS to EC but was reestablished in differentiated components of nonseminomas, distinguished by coexpression of mesodermal markers and loss of OCT4. Subsequent in vitro studies confirmed that 1,25(OH)2D3 (active VD) downregulated NANOG and OCT4 through genomic VDR activation in EC-derived NTera2 cells and, to a lesser extent, in seminoma-derived TCam-2 cells, and up-regulated brachyury, SNAI1, osteocalcin, osteopontin, and fibroblast growth factor 23. To test for a possible therapeutic effect in vivo, NTera2 cells were xenografted into nude mice and treated with 1,25(OH)2D3, which induced down-regulation of pluripotency factors but caused no significant reduction of tumor growth. During NTera2 tumor formation, down-regulation of VDR was observed, resulting in limited responsiveness to cholecalciferol and 1,25(OH)2D3 treatment in vivo. These novel findings show that VD metabolism is involved in the mesodermal transition during differentiation of cancer cells with embryonic stem cell characteristics, which points to a function for VD during early embryonic development and possibly in the pathogenesis of TGCTs.
PMCID: PMC3479852  PMID: 23097629
20.  Phthalate Excretion Pattern and Testicular Function: A Study of 881 Healthy Danish Men 
Environmental Health Perspectives  2012;120(10):1397-1403.
Background: In animals, some phthalates impair male reproductive development and function. Epidemiological studies have reported inconsistent evidence of associations between phthalates and markers of human testicular function.
Objectives: We aimed to provide estimates of the effects of phthalate exposure on reproductive hormone levels and semen quality in healthy men.
Methods: A total of 881 men gave urine, serum, and semen samples. Serum levels of testosterone, estradiol (E2), sex hormone-binding globulin (SHBG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and inhibin-B; semen quality; and urinary concentrations of 14 phthalate metabolites, including metabolites of di(2-ethylhexyl) phthalate (DEHP) and diisononyl phthalate (DiNP), were assessed. The proportions of DEHP and DiNP excreted as their respective primary metabolites [mono(2-ethylhexyl) phthalate (MEHP) and mono-isononyl phthalate (MiNP)] were calculated and expressed as percentages (%MEHP and %MiNP, respectively).
Results: The free androgen index was 15% lower [95% confidence interval (CI): –23, –8%] for men in the highest %MiNP quartile compared to the lowest quartile (p < 0.001) after adjusting for confounders, and 9% lower (95% CI: –16, –1%) in the highest %MEHP quartile (p = 0.02). %MEHP and %MiNP were negatively associated with the ratio of testosterone/LH and testosterone/FSH. %MEHP was negatively associated with total testosterone, free testosterone, and ratio of testosterone/E2. %MiNP was positively associated with SHBG. There was little evidence of associations between urinary phthalate metabolites or sums of phthalates with reproductive hormones or semen quality
Conclusion: Our data suggest that both testosterone production and pituitary–hypothalamic feedback may be compromised in individuals excreting a high proportion of primary metabolites of long-chained phthalates relative to the proportion of secondary metabolites.
doi:10.1289/ehp.1205113
PMCID: PMC3491947  PMID: 22832070
DEHP; DiNP; male reproduction; phthalates; semen quality; testosterone; %MEHP; %MiNP
22.  Human semen quality in the new millennium: a prospective cross-sectional population-based study of 4867 men 
BMJ Open  2012;2(4):e000990.
Objectives
Considerable interest and controversy over a possible decline in semen quality during the 20th century raised concern that semen quality could have reached a critically low level where it might affect human reproduction. The authors therefore initiated a study to assess reproductive health in men from the general population and to monitor changes in semen quality over time.
Design
Cross-sectional study of men from the general Danish population. Inclusion criteria were place of residence in the Copenhagen area, and both the man and his mother being born and raised in Denmark. Men with severe or chronic diseases were not included.
Setting
Danish one-centre study.
Participants
4867 men, median age 19 years, included from 1996 to 2010.
Outcome measures
Semen volume, sperm concentration, total sperm count, sperm motility and sperm morphology.
Results
Only 23% of participants had optimal sperm concentration and sperm morphology. Comparing with historic data of men attending a Copenhagen infertility clinic in the 1940s and men who recently became fathers, these two groups had significantly better semen quality than our study group from the general population. Over the 15 years, median sperm concentration increased from 43 to 48 million/ml (p=0.02) and total sperm count from 132 to 151 million (p=0.001). The median percentage of motile spermatozoa and abnormal spermatozoa were 68% and 93%, and did not change during the study period.
Conclusions
This large prospective study of semen quality among young men of the general population showed an increasing trend in sperm concentration and total sperm count. However, only one in four men had optimal semen quality. In addition, one in four will most likely face a prolonged waiting time to pregnancy if they in the future want to father a child and another 15% are at risk of the need of fertility treatment. Thus, reduced semen quality seems so frequent that it may impair the fertility rates and further increase the demand for assisted reproduction.
Article summary
Article focus
A paper by Carlsen et al 20 years ago (BMJ 1992;305:609–13) raised controversy with evidence of a decline in semen quality, and several studies on semen quality in human populations have followed.
There has been a lack of larger, prospectively collected quality-controlled data on semen quality in the general population.
Key messages
This study brings good and bad news.
Fifteen years monitoring of semen quality in men of the general population indicated a slight increase in both median sperm concentration and total sperm count.
However, still only a fraction of the men (23%) had optimal sperm concentration and sperm morphology, and the median percentage of abnormal spermatozoa was as high as 93% with no sign of improvement during the study period.
Approximately 15% of the men had a sperm concentration at a level that would indicate a high risk of needing future fertility treatment if they want to father a child, and another 27% of the men will be at risk of a prolonged waiting time to pregnancy.
Strengths and limitations of this study
Large prospective study of semen quality among men of the general population unselected with regard to fertility.
Standardised inclusion and investigation procedures.
Lack of historical, directly comparable data.
doi:10.1136/bmjopen-2012-000990
PMCID: PMC3391374  PMID: 22761286
23.  Pentoxifylline Enhances the Radioprotective Properties of γ-Tocotrienol: Differential Effects on the Hematopoietic, Gastrointestinal and Vascular Systems 
Radiation research  2010;175(3):297-306.
The vitamin E analog γ-tocotrienol (GT3) is a potent radioprotector and mitigator. This study was performed to (a) determine whether the efficacy of GT3 can be enhanced by the addition of the phosphodiesterase inhibitor pentoxifylline (PTX) and (b) to obtain information about the mechanism of action. Mice were injected subcutaneously with vehicle, GT3 [400 mg/kg 24 h before total-body irradiation (TBI)], PTX (200 mg/kg 30 min before TBI), or GT3+PTX before being exposed to 8.5–13 Gy TBI. Overall lethality, survival time and intestinal, hematopoietic and vascular injury were assessed. Cytokine levels in the bone marrow microenvironment were measured, and the requirement for endothelial nitric oxide synthase (eNOS) was studied in eNOS-deficient mice. GT3+PTX significantly improved survival compared to GT3 alone and provided full protection against lethality even after exposure to 12.5 Gy. GT3+PTX improved bone marrow CFUs, spleen colony counts and platelet recovery compared to GT3 alone. GT3 and GT3+PTX increased bone marrow plasma G-CSF levels as well as the availability of IL-1α, IL-6 and IL-9 in the early postirradiation phase. GT3 and GT3+PTX were equally effective in ameliorating intestinal injury and vascular peroxynitrite production. Survival studies in eNOS-deficient mice and appropriate controls revealed that eNOS was not required for protection against lethality after TBI. Combined treatment with GT3 and PTX increased postirradiation survival over that with GT3 alone by a mechanism that may depend on induction of hematopoietic stimuli. GT3+PTX did not reduce GI toxicity or vascular oxidative stress compared to GT3 alone. The radioprotective effect of either drug alone or both drugs in combination does not require the presence of eNOS.
doi:10.1667/RR2399.1
PMCID: PMC3115470  PMID: 21388273
24.  Preclinical Evaluation of SOM230 as a Radiation Mitigator in a Mouse Model: Postexposure Time Window and Mechanisms of Action 
Radiation research  2011;175(6):728-735.
The somatostatin analog SOM230 has potent radioprophylactic and radiation mitigating properties that are unrelated to cytoprotection but appear to be due to suppression of secretion of pancreatic enzymes into the intestinal lumen. To determine the maximal postirradiation time window for administration, male CD2F1 mice were exposed to 8.5–11 Gy total-body radiation; SOM230 (0.5, 2 or 5 mg/kg) or vehicle was given by twice daily subcutaneous injections for 14 days, beginning 24–72 h after irradiation, and 30-day animal survival was recorded. The contribution of the gut to systemic cytokine levels was estimated by analyzing plasma samples obtained simultaneously from the portal vein and carotid artery. The effect of SOM230 on cell trypsin secretion was assessed in vitro and intestinal proteolytic activity was measured in vivo. SOM230 was associated with a 40–60% absolute improvement in overall postirradiation survival when treatment was started 48 h after irradiation and even exhibited a statistically significant survival benefit when started at 72 h. SOM230 ameliorated the radiation-induced decrease in chemokine (C-X-C motif) ligand 9 (CXCL9). SOM230 inhibited pancreatic acinar cell trypsin secretion in vitro in a dose-dependent fashion and reduced intraluminal and intestinal tissue proteolytic activity in vivo. SOM230 is an excellent radiation mitigator with a postirradiation time window in excess of 48 h. The mechanism likely involves preservation of intestinal barrier function due to decreased secretion of pancreatic enzymes into the bowel lumen.
PMCID: PMC3118638  PMID: 21529145
25.  Novel Strategies to Ameliorate Radiation Injury: A Possible Role for Tetrahydrobiopterin 
Current Drug Targets  2010;11(11):1366-1374.
Novel pharmacological strategies are urgently needed to prevent or reduce radiation-induced tissue injury. Microvascular injury is a prominent feature of both early and delayed radiation injury. Radiation-induced endothelial dysfunction is believed to play a key role in the pathogenesis of post-irradiation tissue injury. Hence, strategies that could prevent or improve endothelial malfunction are expected to ameliorate the severity of radiation injury. This review focuses on the therapeutic potential of the nitric oxide synthase (NOS) cofactor 5,6,7,8-tetrahydrobiopterin (BH4) as an agent to reduce radiation toxicity. BH4 is an essential cofactor for all NOS enzymes and a critical determinant of NOS function. Inadequate availability of BH4 leads to uncoupling of the NOS enzyme. In an uncoupled state, NOS produces the highly oxidative radicals superoxide and peroxynitrite at the cost of NO. Under conditions of oxidative stress, such as after radiation exposure, BH4 availability might be reduced due to the rapid oxidation of BH4 to 7,8-dihydrobiopterin (7,8-BH2). As a result, free radical–induced BH4 insufficiency may increase the oxidative burden and hamper NO-dependent endothelial function. Given the growing evidence that BH4 depletion and subsequent endothelial NOS uncoupling play a major role in the pathogenesis of endothelial dysfunction in various diseases, there is substantial reason to believe that improving post-irradiation BH4 availability, by either supplementation with it or modulation of its metabolism, might be a novel strategy to reduce radiation-induced endothelial dysfunction and subsequent tissue injury.
PMCID: PMC3311028  PMID: 20583982
Radiation injuries; radioprotection; endothelial dysfunction; nitric oxide synthase; tetrahydrobiopterin; HMG-CoA reductase; γ-tocotrienol

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