Soluble Dietary Fiber Ameliorates Radiation-Induced Intestinal Epithelial-to-Mesenchymal Transition and Fibrosis.

BACKGROUND: Intestinal fibrosis is a late complication of pelvic radiotherapy. Epithelial-to-mesenchymal transition (EMT) plays an important role in tissue fibrosis. The aim of this study was to examine the effect of soluble dietary fiber on radiation-induced intestinal EMT and fibrosis in a mouse model. MATERIALS AND METHODS: Apple pectin (4% wt/wt in drinking water) was administered to wild-type and pVillin-Cre-EGFP transgenic mice with intestinal fibrosis induced by a single dose of abdominal irradiation of 10 Gy. The effects of pectin on intestinal EMT and fibrosis, gut microbiota, and short-chain fatty acid (SCFA) concentration were evaluated. RESULTS: Intestinal fibrosis in late radiation enteropathy showed increased submucosal thickness and subepithelial collagen deposition. Enhanced green fluorescent protein (EGFP)+/vimentin+ and EGFP+/α-smooth muscle actin (SMA)+ coexpressing cells were most clearly observed at 2 weeks after irradiation and gradually decreased at 4 and 12 weeks. Pectin significantly attenuated the thickness of submucosa and collagen deposition at 12 weeks (24.3 vs 27.6 µm in the pectin + radiation-treated group compared with radiation-alone group, respectively, P < .05; 69.0% vs 57.1%, P < .001) and ameliorated EMT at 2 and 4 weeks. Pectin also modulated the intestinal microbiota composition and increased the luminal SCFA concentration. CONCLUSION: The soluble dietary fiber pectin protected the terminal ileum against radiation-induced fibrosis. This effect might be mediated by altered SCFA concentration in the intestinal lumen and reduced EMT in the ileal epithelium.

Ciprofloxacin Therapy Results in Mitigation of ATP Loss after Irradiation Combined with Wound Trauma: Preservation of Pyruvate Dehydrogenase and Inhibition of Pyruvate Dehydrogenase Kinase 1.

Ionizing radiation exposure combined with wound injury increases animal mortalities than ionizing radiation exposure alone. Ciprofloxacin (CIP) is in the fluroquinolone family of synthetic antibiotic that are available from the strategic national stockpile for emergency use and is known to inhibit bacterial sepsis. The purpose of this study was to evaluate the efficacy of ciprofloxacin as a countermeasure to combined injury mortality and determine the signaling proteins involved in energy machinery. B6D2F1/J female mice were randomly assigned to receive either 9.75 Gy irradiation with Co-60 gamma rays followed by skin wounding (combined injury; CI) or sham procedure (sham). Either ciprofloxacin (90 mg/kg/day) or vehicle (VEH) (water) was administered orally to these mice 2 h after wounding and thereafter daily for 10 days. Determination of tissue adenosine triphosphate (ATP) was conducted, and immunoblotting for signaling proteins involved in ATP machinery was performed. Combined injury resulted in 60% survival after 10 days compared to 100% survival in the sham group. Furthermore, combined injury caused significant reductions of ATP concentrations in ileum, pancreas, brain, spleen, kidney and lung (-25% to -95%) compared to the sham group. Ciprofloxacin administration after combined injury resulted in 100% survival and inhibited reductions in ileum and kidney ATP production. Ileum protein levels of heat-shock protein 70 kDa (HSP-70, a chaperone protein involved in ATP synthesis) and pyruvate dehydrogenase (PDH, an enzyme complex crucial to conversion of pyruvate to acetyl CoA for entrance into TCA cycle) were significantly lower in the CI group (vs. sham group). Using immunoprecipitation and immunoblotting, HSP-70-PDH complex was found to be present in the ileum tissue of CI mice treated with ciprofloxacin. Furthermore, phosphorylation of serine residues of PDH resulting in inactivating PDH enzymatic activity, which occurred after combined injury, was inhibited with ciprofloxacin treatment, thus enabling PDH to increase ATP production. Increased ileum levels of pyruvate dehydrogenase kinase 1 protein (PDK1, an enzyme responsible for PDH phosphorylation) after combined injury were also prevented by ciprofloxacin treatment. Taken together, these data suggest that ciprofloxacin oral administration after combined injury had a role in sustained ileum ATP levels, and may have acted through preservation of PDH by HSP-70 and inhibition of PDK1. These molecular changes in the ileum are simply one of a host of mechanisms working in concert with one another by which ciprofloxacin treatment mitigates body weight loss and drastically enhances subsequent survival after combined injury. To this end, our findings indicate that oral treatment of ciprofloxacin is a valuable therapeutic treatment after irradiation with combined injury and warrants further analyses to elucidate the precise mechanisms involved.

Bile acid malabsorption after pelvic and prostate intensity modulated radiation therapy: an uncommon but treatable condition.

PURPOSE: Intensity modulated radiation therapy (IMRT) is a significant therapeutic advance in prostate cancer, allowing increased tumor dose delivery and increased sparing of normal tissues. IMRT planning uses strict dose constraints to nearby organs to limit toxicity. Bile acid malabsorption (BAM) is a treatable disorder of the terminal ileum (TI) that presents with symptoms similar to radiation therapy toxicity. It has not been described in patients receiving RT for prostate cancer in the contemporary era. We describe new-onset BAM in men after IMRT for prostate cancer. METHODS AND MATERIALS: Diagnosis of new-onset BAM was established after typical symptoms developed, selenium-75 homocholic acid taurine (SeHCAT) scanning showed 7-day retention of <15%, and patients' symptoms unequivocally responded to a bile acid sequestrant. The TI was identified on the original radiation therapy plan, and the radiation dose delivered was calculated and compared with accepted dose-volume constraints. RESULTS: Five of 423 men treated in a prospective series of high-dose prostate and pelvic IMRT were identified with new onset BAM (median age, 65 years old). All reported having normal bowel habits before RT. The volume of TI ranged from 26-141 cc. The radiation dose received by the TI varied between 11.4 Gy and 62.1 Gy (uncorrected). Three of 5 patients had TI treated in excess of 45 Gy (equivalent dose calculated in 2-Gy fractions, using an α/ß ratio of 3) with volumes ranging from 1.6 cc-49.0 cc. One patient had mild BAM (SeHCAT retention, 10%-15%), 2 had moderate BAM (SeHCAT retention, 5%-10%), and 2 had severe BAM (SeHCAT retention, <5%). The 3 patients whose TI received ≥45 Gy developed moderate to severe BAM, whereas those whose TI received <45 Gy had only mild to moderate BAM. CONCLUSIONS: Radiation delivered to the TI during IMRT may cause BAM. Identification of the TI from unenhanced RT planning computed tomography scans is difficult and may impede accurate dosimetric evaluation. Thorough toxicity assessment and close liaison between oncologist and gastroenterologist allow timely diagnosis and treatment.

Successful mitigation of delayed intestinal radiation injury using pravastatin is not associated with acute injury improvement or tumor protection.

PURPOSE: To investigate whether pravastatin mitigates delayed radiation-induced enteropathy in rats, by focusing on the effects of pravastatin on acute cell death and fibrosis according to connective tissue growth factor (CTGF) expression and collagen inhibition. METHODS AND MATERIALS: Mitigation of delayed radiation-induced enteropathy was investigated in rats using pravastatin administered in drinking water (30 mg/kg/day) 3 days before and 14 days after irradiation. The ileum was irradiated locally after surgical exteriorization (X-rays, 19 Gy). Acute apoptosis, acute and late histologic alterations, and late CTGF and collagen deposition were monitored by semiquantitative immunohistochemistry and colorimetric staining (6 h, 3 days, 14 days, 15 weeks, and 26 weeks after irradiation). Pravastatin antitumor action was studied in HT-29, HeLa, and PC-3 cells by clonogenic cell survival assays and tumor growth delay experiments. RESULTS: Pravastatin improved delayed radiation enteropathy in rats, whereas its benefit in acute and subacute injury remained limited (6 h, 3 days, and 14 days after irradiation). Delayed structural improvement was associated with decreased CTGF and collagen deposition but seemed unrelated to acute damage. Indeed, the early apoptotic index increased, and severe subacute structural damage occurred. Pravastatin elicited a differential effect, protecting normal intestine but not tumors from radiation injury. CONCLUSION: Pravastatin provides effective protection against delayed radiation enteropathy without interfering with the primary antitumor action of radiotherapy, suggesting that clinical transfer is feasible.

Ginkgo biloba extract protects against ionizing radiation-induced oxidative organ damage in rats.

The present study was designed to determine the possible protective effects of Ginkgo biloba extract (EGb) against oxidative organ damage induced by irradiation (IR). Sprague-Dawley rats were exposed to whole-body IR (800 cGy) after a 15-day pretreatment with either saline or EGb (50 mg/kg/day), intraperitoneally, and treatments were repeated immediately after the IR. Then the rats were decapitated at either 6 h or 72 h after IR, where EGb or saline injections were repeated once daily. Lung, liver, kidney and ileum samples were obtained for the determination of malondialdehyde, glutathione levels, myeloperoxidase activity and collagen contents, while oxidant-induced DNA fragmentation was evaluated in the ileal tissues. All tissues were also examined microscopically and assayed for the production of reactive oxidants using chemiluminescence (CL). Lactate dehydrogenase (LDH)-an indicator of tissue damage and TNF-alpha were assayed in serum samples. In the saline-treated irradiation groups, glutathione levels were decreased significantly, while the malondialdehyde levels, myeloperoxidase activity and collagen content were increased in the tissues (p < 0.01-0.001), which were in parallel with the increases in luminol and lucigenin CL values. In the EGb treated-IR groups, all of these oxidant responses were prevented significantly (p < 0.05-0.01). LDH and TNF-alpha levels, which were increased significantly (p < 0.01-0.001) following IR, were decreased (p < 0.05-0.001) with EGb treatment. In conclusion, the present data demonstrate that EGb, through its free radical scavenging and antioxidant properties, attenuates irradiation-induced oxidative organ injury, suggesting that EGb may have a potential benefit in enhancing the success of radiotherapy.

Melatonin ameliorates ionizing radiation-induced oxidative organ damage in rats.

This study was designed to study the effects of the potential radioprotective properties of pharmacological doses of melatonin against organ damage induced by whole-body irradiation (IR) in rats. A total of 32 male Sprague-Dawley rats were exposed to irradiation performed with a LINAC producing 6 MV photons at a focus 100 cm distant from the skin. Under ketamine anaesthesia, each rat received a single whole-body dose of 800 cGy. Immediately before and after IR, rats were treated with either saline or melatonin (20 mg/kg and 10 mg/kg, i.p.) and decapitated at 12-h after exposure to irradiation. Another group of rats was followed for 72-h after IR, where melatonin (10 mg/kg, i.p.) injections were repeated once daily. Tissue levels of malondialdehyde (MDA)--an index of lipid peroxidation--, glutathione (GSH)--a key to antioxidant--and myeloperoxidase (MPO) activity--an index of neutrophil infiltration--were estimated in liver, lung, colon and intestinal tissues. The results demonstrate that both 12-h and 72-h following IR, tissue levels of MDA were elevated (p<0.05-0.001), while GSH levels were reduced (p<0.05-0.001) in all organs. On the other hand, melatonin, reduced the levels of MDA and increased the GSH levels significantly, (p<0.05-0.001). MPO activity was increased significantly in the colonic tissue at the both 12-h and 72-h, and in the hepatic tissue at the 72-h following IR, which were reduced by melatonin (p<0.01-0.001). In the lung tissue enzyme activity was decreased at 72nd h of post-irradiation. In conclusion, the increase in MDA levels and MPO activity and the concomitant decrease in GSH levels demonstrate the role of oxidative mechanisms in irradiation-induced tissue damage, and melatonin, by its free radical scavenging and antioxidant properties, ameliorates irradiation-induced organ injury. Thus, supplementing cancer patients with adjuvant therapy of melatonin may have some benefit for successful radiotherapy.

The effect of selenium and/or vitamin E treatments on radiation-induced intestinal injury in rats.

Cytotoxic effects of ionizing radiation on gastrointestinal epithelium may be related to oxidative stress. In this study, we wanted to investigate the effects of selenium, vitamin E and selenium plus vitamin E pretreatments prior to whole abdominal irradiation on intestinal injury. Irradiation caused increased lipid peroxide and decreased GSH levels in the intestine. Intestinal superoxide dismutase and glutathione peroxidase activities were increased, but glutathione transferase activity decreased following irradiation. Selenium and/or vitamin E pretreatments ameliorated these disturbances in prooxidant-antioxidant balance. This amelioriation has been verified with histopathological findings. These results indicate that antioxidant pretreatments prior to irradiation may have some beneficial effects against irradiation-induced intestinal injury.

Protective effect of vitamin A on acute radiation injury in the small intestine.

The objective of this study was to examine the influence of vitamin A on the development of early radiation-induced reactions in the rat small intestine. The early effects of intraoperative gamma-radiation on the small bowel utilizing the terminal ileum of Sprague-Dawley rats and the protective effect of supplemental vitamin A on acute radiation injury were investigated. Three groups were included in the study: group I (10 rats) was the surgical control group; group II (13 rats) underwent only intraoperative irradiation; and group III (10 rats) was the vitamin A plus irradiation group. Exteriorized terminal ileal segments of groups II and III were exposed to a single fraction of 20 Gy of intraoperative gamma-irradiation. On the seventh postoperative day, terminal ileal segments of all rats were resected and histopathologically evaluated for ulceration, enteritis cystica profunda, atypical epithelial regeneration, fibrosis, vascular sclerosis, and inflammatory process. Although none of the above findings were present in the surgical control group, group III rats experienced less severe effects than group II rats. The results suggest the early side effects of radiation may be prevented by vitamin A supplementation.

Early effects on the morphology of mouse small intestine of single or combined modality treatment with hyperthermia and X-irradiation.

This study describes the effects of hyperthermia and X-irradiation on the morphological appearance of normal, at risk tissues in the ileum of the mouse. The early morphological effects 1 day after a combined modality treatment are compared with those due to either hyperthermia or X-irradiation given alone. The response was assessed qualitatively and semiquantitatively using scanning electron microscopy and a villous scoring technique. Early post-irradiation effects on topography did not differ significantly from those observed after small intestine exteriorisation without treatment. The villous scores for the combined modality treatments reflected greater damage than would be expected from the sum of villous scores for each modality treatment on its own. This suggests that the combined modality treatment had a synergistic or enhancing effect. A 4 hour time interval between the two treatments did not seem to reduce the enhancing effect. Further studies are required to investigate the effects of fractionated combined treatment.