5-Fluorouracil-Induced Changes of Intestinal Integrity Biomarkers in BALB/C Mice

Background: Intestinal mucositis is a most frequently occurring toxicity in cancer chemotherapy, and consequent malnutrition reduces tolerance to cancer therapies. Therefore it is important to lessen the severity of mucotitis and to develop complementary agents capable of reducing mucotitis-related symptoms. This study was conducted to determine 5-fluorouracil (5-FU) induced intestinal damage to understand intestinal damages due to chemotherapy and to provide information on biomarkers which can be used to screen complementary agents in future studies. Methods: BALB/c mice were divided into three experimental groups and subjected to the intraperitoneal injection of either 100 mg/kg or 200 mg/kg of 5-FU. The third group was used as PBS controls. Body weights and the consistency of the stools were recorded every day, and the animals were sacrificed on the 7th day post 5-FU administration. The expressions of intestinal tight junction proteins and mRNAs of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) were determined. Results: The body weight of the animals treated with 5-FU was significantly decreased in a dose-dependent manner. However, mice given 100 mg/kg 5-FU rapidly recovered the original body weight. Symptom of diarrhea was also more severe in 200 mg/kg 5-FU treated group than that of the 100 mg/kg 5-FU treated animals. The expressions of occludin and claudin-1, not ZO-1 protein expressions in 200 mg/kg 5-FU treated animals were significantly reduced compared to those of the control group or 100 mg/kg 5-FU group. The expression of Nuclear factor-kappa B p65 (NF-κB p65) protein and TNF-α mRNA were significantly higher in 5-FU treated group compared to those of control group. No difference was observed with IL-1β expression. Conclusions: These results suggested that selected tight junction proteins and inflammatory cytokines are related to 5-FU induced mucositis, and thereby can be used as targets of developing complementary agents.


INTRODUCTION
Gastrointestinal mucositis is a common side effect of anticancer chemotherapy that involves the small intestines. Not only does mucositis decrease the quality of life in most cancer patients because of its associated intense pain, it is also a high-risk factor for hematosepsis with neutropenia and malnutrition. This association, thus, renders mucositis a clinically important disease. Moreover, this specific side effect does not allow appropriate chemotherapy and radiotherapy, and becomes an obstacle that delays the administration of effective cancer treatment. 1 5-Fluorouracil (5-FU) is an anticancer drug that is widely used in the treatment of colorectal cancer, and it possesses a chemical structure similar to that of uracil and thymine. 2 A major part of it (more than 80%) is catabolized by hepatic dihydropyrimidine dehyrogenase (DPD), whereas the rest induces cell death by inhibiting RNA and DNA syntheses through fluorodeoxyuridine monophosphate (FdUMP) and fluorouridine triphosphate (FUTP). 3,4 The major side effects of 5-FU include myelosuppression, diarrhea, cardiotoxicity, dermatitis, and mucositis. 5,6 Of these, gastrointestinal mucositis has been reported in approximately 80% of patients who have received cancer treatment by using

Sample collection and pretreatment
Once the treatment period was completed, the mice were

Diarrhea assessment
The presence or absence and degree of diarrhea were determined based on the standards established for the diarrhea assessment. 8 The diarrhea score was assessed  The extracted proteins were quantified using a Protein   quantification values and 2 -ΔΔCt were calibrated using β -actin, which is a housekeeping gene. Each experiment was performed in triplicate.

Statistical analysis
For the results of all experimental analyses, means and standard deviation in each group were calculated using a SAS program. After performing one-way ANOVA, statistical significance of the means in each group was tested using Duncan's multiple range test, at a significance level of α= 0.05.

Body weight changes and main organ weight
Body weight changes in the experimental animals are shown in Fig. 2. Compared to the control group, the extent of body weight reduction in the experimental groups manifested significant differences from Day 1 after the 5-FU administration ( Fig. 2A, P＜0.05). Significantly higher reduction in the body weight was observed in the group administered with 200 mg/kg of 5-FU compared to the 100 mg/kg group (Fig. 2A). The weights of the liver and kidney and the lengths of the small and large intestines are presented in Table 2. The weight of the spleen significantly decreased in the 5-FU treated experimental groups (P＜0.05). No significant differences in the weight of the liver and lengths of the small and large intestines were observed.

Diarrhea assessment
The  responses. NF-κB protein expression in the mucous of the small intestine was significantly higher in both the groups compared to that in the control (Fig. 4, P＜0.005). The expression level was slightly lower in the group in which high concentration of 5-FU was used compared to that in the low concentration (Fig. 4, P＜0.05).  permeability is shown to result in the inflow of endotoxins, various pathogenic microorganisms, and varied high molecular substances. 19 A previous study suggested that occludin knockout mice exhibited not only chronic inflammation and hyperplasia of the gastric epithelium but abnormal changes in testicule and salivary gland suggesting more complex role of occludin. 20 A previous study suggested that hydrogen peroxide induced TJ disruption is mediated by a tyrosine kinase dependent mechanism. 21 Claudin is a protein structurally discriminated from occuldin. Claudins are suggested as the backbone of TJ proteins, and the claudin knockout mice revealed abnormal barrier formation and paracellular permselectivity. 22 Our results suggest that 5-FU treatment significantly reduces intestinal membrane TJ protein expression possibly exhibiting stuructural and functional defects. In the case of ZO-1, which is one of the TJ proteins, no significant intergroup differences were observed. Such discrepancies in the TJ proteins may be attributable to their different interactions with other membrane compartments including actin. Although ZO-1 proteins are known to mediate early TJ protein assembly, functional role of ZO-1 has not been clearly demonstrated. Also, a recent report showed that normal TJ structures were formed with ZO-1 deficient cells suggesting further studies on the role of ZO-1 in TJ proteins. 23 Since our results indicate claudins and occuldin expressions are dramatically reduced by 5-FU treatment, it is presumable that severe functional defects are induced by 5-FU treatment and therefore appropriate interventions towards there TJ proteins may alleviate chemotherapy-induced mucositis.

TNF-α and
In conclusion, 5-FU treated animals revealed significant body weight loss in a dose-dependent manner. Clear and significant TJ protein losses are observed suggesting appropriate intervention towards these targets may alleviate chemotherapy-associated mucositis.