Journal of Cancer Research and Therapeutics Medknow Publications on behalf of the Association of Radiation Oncologists of India (AROI) ISSN: 0973-1482 EISSN: 1998-4138 Vol. 7, Num. 2, 2011, pp. 152-156

Journal of Cancer Research and Therapeutics, Vol. 7, No. 2, April-June, 2011, pp. 152-156

Original Article

The effect of a flavonoid fractions diosmin + hesperidin on radiation-induced acute proctitis in a rat model

Atakan Sezer¹, Ufuk Usta², Zafer Kocak³, Mehmet Ali Yagci¹

¹ Department of General Surgery, Faculty of Medicine, Trakya University, Turkey
² Department of Pathology, Faculty of Medicine, Trakya University, Turkey
³ Department of Radiation Oncology, Faculty of Medicine, Trakya University, Turkey
Correspondence Address: Atakan Sezer, Department of General Surgery, Trakya University, Edirne 22030, Turkey, atakansezer@hotmail.com

Code Number: cr11036

PMID: 21768702
DOI: 10.4103/0973-1482.82927

Abstract

Keywords: Acute radiation proctitis, Daflon, flavonoid fractions, radiotherapy

Introduction

Radiotherapy for malignant pelvic disease is often mandatory. Acute radiation proctitis is a complication of this treatment which present with bleeding, mucus discharge, urgency, discomfort, anal pain, and incontinence. ^[1] The complication rates reaches to 10%−15% among patients treated with pelvic irradiation. ^[2] Inflammation, edema, epithelial degeneration, ulceration, crypt disintegration, mucosal edema, absent mitosis, and crypt abscesses are the histopathological alterations caused by radiation exposed to rectum. ^[3],[4]

The overall management of radiation proctitis has been unsatisfactory, as only a few experimental or clinical treatment modalities were experienced consist of topical butyrate, steroids, sucralfates, short-chain fatty acids, hyperbaric oxygen, mesalazine, formalin, and anti-inflammatory agents. ^{[5],[6],[7],[8],[9],[10],[11],[12]} Salt-water baths, stool conditioners, and analgesics may provide symptomatic relief.

Flavonoids are a group of plant extracts with structural, biochemical, and pharmacological usage. The anti-inflammatory, immunomodulatory, and anti-oxidant specialties are the multifactorial effects of flavonoids in vivo and in vitro. ^[13],[14] The present experimental study was thus aimed at assessing the efficacy of micronized, purified, flavonoid fractions (Daflon, 500 mg; Servier, Turkey) which is cheap and easy attainable drug, in the management of acute radiation proctitis in a rat model.

Materials and Methods

The study was performed at the laboratory of "The Unite of the Laboratory Animals for Experimental Studies of Trakya University Medical School" in accordance with the guidelines for the care and use of the laboratory animals established by the "Animal Ethics committee of Trakya University" following the approval of the design by the "Animal Ethics committee of Trakya University". Thirty four female animals Sprague-Dawley rats (weights: 250 ± 30 g and ages: between 11 and 13 weeks) were divided into four groups. The animal room was maintained at a temperature of 22 ± 2 ⁰ C and a relative humidity of 55 ± 15% with a 12-h light-dark cycle. Tap water was freely available throughout the acclimatization and experiment periods.

The rats in group 1 received Daflon and underwent irradiation (10 rats). The rats in group 2 received no Daflon and underwent irradiation (eight rats). The rats in group 3 received Daflon and underwent sham irradiation (eight rats). The rats in group 4 received no Daflon and underwent sham irradiation (eight rats).

Daflon (Micronized, purified, flavonoid fraction 500 mg; Servier, Turkey) was commercially purchased. An emulsion (500 mg Daflon in 10 ml distilled water) were prepared and 100 mg/kg/day which corresponds to 0.5 ml Daflon emulsion was administered via an orogastric feeding tube to the rats in groups 1 and 3 starting from one day prior to irradiation until the euthanasia day (day 15 following irradiation). For the rats in groups 2 and 4, same feeding protocol with 0.9% sodium chloride was performed.

Each rat was anesthetized using 5 mg/kg of xylazine (Rompun, Bayer Türk Kimya Sanayi Limited Şirketi, İstanbul, Turkey) and 30 mg/kg of ketamine hydrochloride (Ketalar, Pfizer İlaçları Limited Şirketi, İstanbul) intramuscularly prior to irradiation. Then, each rat was restrained and taped by the tail and legs on an acryl plate at a supine position. Lead shielding (5 half value layer) was used to cover the rat except for a 3 × 4 cm area of lower pelvis containing 2 cm length of rectum in the middle of the field. Irradiation was delivered on a cobalt-60 unit with the distance of 80 cm from the source to surface using a single fraction of 17.5 Gy defined at a depth of 1 cm through an anterior portal.

Prior to euthanasia on day 15 following irradiation or sham irradiation, euthanasia was performed by cardiac exsanguination under a surgical plane of ketamine and xylazine anesthesia. The rectum specimens excised for histopathological evaluation.

Representative sections of all rectal segments were embedded into paraffin after fixation of the tissues in 10% neutral buffered formalin. Five micrometer sections were obtained from each paraffin block and stained with hematoxylin and eosin. Slides were examined by the same pathologist under a light microscope (Nikon Eclipse E600) two times in a blinded manner. Inflammatory infiltrate in lamina propria (Inflammation), infiltration of crypt epithelium by leucocytes (cryptitis), distortion of the crypts (Crypt distortion) and regenerative/reparative atypia of the epithelial cells (reactive atypia) were examined and were degreed from 0 to 3 according to the severity of the lesions, where; 0 means no lesion, 1 means mild lesion, 2 means moderate lesion and 3 means severe lesion [3]. Crypt abscess was noted as positive or negative for each slide. Thicknesses of mucosa and muscularis propria were measured from their thinnest portions across the diameters of the lumina of the bowels by using an image analyzer attached to a video-microscope (Zeiss Axioplan 2 imaging).

The numeric results were expressed as mean and categorical results were expressed as a number. The Chi-square test was used to compare the differences of categorical variables (inflammation, cryptitis, crypt distortion, and reactive atypia) among the groups. Mean values for mucosa and muscular wall thickness were compared using the one-way analysis of variance (ANOVA). A P-value <0.05 was considered as statistically significant. Statistica 7.0 (StatSoft Inc. Tulsa, OK, USA) statistical software was used for statistical analyses.

Results

No mortality was observed in groups 1, 2, and 4. Two rats died within 2 h after the procedure in Group 3 and autopsy was performed to clarify the deaths. The main causes of the deaths were iatrogenic tracheal intubations and respiratory insufficiency due to aspiration of Daflon during the administration via feeding tube. The rectums of these rats were not available for the histopathological evaluation.

The histopathological evaluation of the rectum by groups is shown in [Table - 1]. When compared to group 2, the rats of group 1 showed less glandular distortion and less mucosal inflammation with less infiltration of the crypt epithelia by the inflammatory cells [Figure - 1] and [Figure - 2]. The decrease of the inflammation in the treatment group was statistically significant (P < 0.001).

A statistically significant increase in all parameters but muscular wall thickness was observed for the rats in group 2 as compared to the group 3 and 4. There was severe mucosal inflammation in the bowels of rats of group 2 [Figure - 3]. The inflammation was mainly in the lamina propria, infiltrating the crypt epithelia leading to cryptitis and severe crypt abscesses. The mucosal glands revealed severe distortion and the epithelial cells revealed severe reactive/regenerative atypia with nuclear widening, hyperchromasia, irregularity, pleomorphism with prominent nucleoli and loss of cytoplasmic mucin [Figure - 4]. The histological appearances of the rats of group 3 and group 4 were almost similar and there was no mucosal inflammatory infiltration in the bowels of those groups of rats [Figure - 5] and [Figure - 6].

Discussion

Histopathological alterations in rectum during pelvic irradiation consist of hyperemia, edema, extensive inflammatory cell infiltration in mucosa, cryptitis, and crypt abscess. ^{[3],[15],[16]} In the current report, a prominent mucosal inflammation with severe cryptitis and crypt abscesses was observed for the rats in group 2 (underwent irradiation only). There were also severe distortions and crybriformity in the glandular structures in the bowel wall. Increased thickness of the mucosal wall compared to the muscularis propria wall was also observed as a radiation effects in group 2. Reactive cytological atypia characterized by nuclear pleomorphism, widening and irregularity of prominent nucleoli, and also loss of cytoplasmic mucin were other histopathological alterations noted in group 2.

Flavonoids are phenolic compounds characterized by their diaryl nucleus. They are structurally similar to steroid hormones. ^[17] Daflon as a flavonoid drug is a micronized purified flavonoid fraction consisting of 90% diosmin and 10% hesperidin. ^[14] The most common indications of Daflon are venous insufficiency and hemorrhoidal diseases in which many studies have demonstrated the potential effects on perivascular edema and inflammatory disease. ^[14],[18]

The inflammatory process plays a crucial role in the pathogenesis and progression of irradiation-induced cellular damage. Arachidonic acid metabolism results in products that play a role in the inflammatory reaction. ^[19] The improvement of histological injury consist of morphologic changes in the superficial epithelium, glands, and stroma of the lamina propria in the rectal mucosa during pelvic radiotherapy may be achieved by inhibition of pro or inflammatory processes. Flavonoids have been found to reduce the activity of key enzymes of the arachidonic acid metabolism. ^[14],[20] Moreover, Daflon as a flavonoid drug has a strong anti inflammatory effect and decreases the release of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule (ICAM-1) which leads to inhibition of leukocyte activation, adhesion, and migration. In the current study, there was a statistically significant decrease in inflammation [Figure - 1] and [Figure - 3] for the rats received Daflon (group 1) as compared to the rats received no Daflon (group 2).

Radiation therapy induces oxidative stress and it has been shown that free oxygen radicals have an essential role in radiation-induced normal tissue toxicity and in inflammation. The decrease in antioxidant enzyme levels may prevents damage of irradiation to normal tissues and decreases the inflammation. ^[21] Flavonoids have been studied for their antioxidant and radical scavenger properties and it has been reported that Daflon is a powerful scavenger of antioxidant enzymes such as superoxide, singlet oxygen and hydroxyl radicals. ^[21] Our results show that mucosal damage caused by irradiation may be diminished by using Daflon but we do not know that this effect may be due to the decrease to the levels of free oxygenated radicals.

There is no available therapy has, as yet, proven effective to treat acute radiation proctitis. In a randomized, double-blind study of Vernia et al., ^[6] 20 patients presented with acute radiation proctitis treated with topical sodium butyrate for three weeks. Clinical, endoscopic, and histological findings demonstrate the effectiveness of sodium butyrate in acute radiation proctitis. Kochhar et al. ^[22] compared oral sulfasalazine plus rectal steroids versus rectal sucralfate in 37 patients in a prospective, randomized, double-blind controlled trial. While the anti-inflammatory group showed clinical improvement compared sucralfate group, endoscopic findings were superior compared to the anti-inflammatory group. Rougier et al., ^[23] worked on the efficiency of local administration of two types of corticosteroid consist of betamethasone enema and hydrocortisone acetate mousse. Over the 4 weeks of treatment the endoscopic appearance improved in a greater proportion of the hydrocortisone group rather than the betamethasone group. Short-chain fatty acids, hyperbaric oxygen, mesalazine, formalin, anti-inflammatory agents, and amiphostine were experienced in the treatment or prevention of radiation proctitis but none of them concluded in a satisfactory result. ^{[9],[10],[11],[12],[23],[24]}

Conclusion

Administration of a dose of 100 mg/kg of the flavonoid fractions diosmin + hesperidin resulted in decreased morphologic changes and inflammatory response. Therefore this drug, which is cheap and easy attainable, may have some protective effects against radiation-induced acute proctitis. Further experimental and clinical studies are needed to address the role of Daflon on radiation-induced bowel toxicity.

Acknowledgments

A.S. was involved in the study design, data collection, and writing and editing all aspects of this manuscript. U.U., Z.K., and M.A.Y. were involved in the study design and editing this manuscript. All of the authors have read and approved the final version of this manuscript.

References

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