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research-article2014
PENXXX10.1177/0148607114567508Journal of Parenteral and Enteral NutritionAntunes et al
Brief Communication
Pretreatment With L-Citrulline Positively Affects the Mucosal Architecture and Permeability of the Small Intestine in a Murine Mucositis Model
Journal of Parenteral and Enteral Nutrition Volume XX Number X Month 201X 1–8 © 2015 American Society for Parenteral and Enteral Nutrition DOI: 10.1177/0148607114567508 jpen.sagepub.com hosted at online.sagepub.com
Maísa Mota Antunes, MSc1; Paola Caroline Lacerda Leocádio, MSc1; Lílian Gonçalves Teixeira, PhD2; Alda Jusceline Leonel, PhD3; Denise Carmona Cara, PhD4; Gustavo Batista Menezes, PhD4; Simone de Vasconcelos Generoso, PhD5; Valbert Nascimento Cardoso, PhD6; Jacqueline Isaura Alvarez-Leite, PhD3; and Maria Isabel Toulson Davisson Correia, PhD7
Abstract Background: Mucositis is a common complication in patients undergoing radiotherapy and chemotherapy. It is associated with pain, poor quality of life, and malnutrition, leading to an increased number of hospital admissions and prolonged hospitalization. The use of immunonutrients may be an alternative treatment option, which may help to improve patient outcome. Objective: Here we assessed the impact of L-citrulline (CIT) on a murine model of 5-fluorouracil (5FU)–induced mucositis. Methods: Swiss male mice were randomized into 4 groups: control, CIT, 5FU, and 5FU+CIT. Mice were fed with commercial chow and supplemented with an oral solution of alanine (control and 5FU groups) or CIT (CIT and 5FU+CIT groups). On the seventh day, mice received intraperitoneal phosphatebuffered saline or 5FU (200 mg/kg, single dose) to induce mucositis. On the 10th day, mice were euthanized, and the blood and small intestines were harvested. Body weight, morphology, histopathology score (hematoxylin and eosin) of the small intestine (from 0–12), myeloperoxidase activity, oxidative stress level, and intestinal permeability were assessed. Results: We observed significant weight loss after the administration of 5FU in both treated and control animals. CIT administration contributed to a partial recovery of the mucosal architecture as well as an intermediate reduction of the histopathologic score, and functional intestinal permeability was partially rescued. Conclusions: CIT administration attenuated 5FU-mediated damage to the mucosal architecture of the small intestine, decreasing the size of the injured areas and promoting decreased intestinal permeability. (JPEN J Parenter Enteral Nutr. XXXX;xx:xx-xx)
Keywords L-citrulline; mucositis; 5FU; intestinal permeability
Clinical Relevancy Statement Mucositis affects 40% of patients undergoing chemotherapy and radiotherapy. Pain, nausea, and diarrhea are common symptoms, which can worsen the nutrition status of the patients, with a direct impact on their quality of life. The use of immunonutrients has been reported to positively affect the recovery from intestinal lesions. In this regard, L-citrulline, a non-proteinogenic neutral amino acid, used in this study may be a strategy to alleviate the symptoms of mucositis, with improvement of nutrition status and decreased length of hospital stay.
Introduction Patients with cancer are frequently undernourished, and chemotherapy and radiotherapy increase the risk of mucositis.1 Pain, nausea, vomiting, and diarrhea are common symptoms in these patients, which can worsen their nutrition status and directly affect their quality of life,2 causing longer hospital stays and higher costs.3 Chemotherapy with 5-fluorouracil (5FU), a drug that is still commonly used in oncologic treatment, inhibits DNA synthesis, resulting in increased apoptosis and decreased cell proliferation, leading to villus and crypt atrophy with concomitant
From the 1Departamento de Ciência de Alimentos, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte–MG, Brazil; 2Departamento de Ciência dos Alimentos, Setor de Nutrição, Universidade Federal de Lavras, Lavras–MG, Brazil; 3Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte–MG, Brazil; 4Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte–MG, Brazil; 5Departamento de Nutrição, Escola de Enfermagem, Universidade Federal de Minas Gerais, Belo Horizonte–MG, Brazil; 6Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte–MG, Brazil; and 7Departamento de Cirurgia, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte–MG, Brazil. Financial disclosure: This work was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Pró-Reitoria de Pesquisa da Universidade Federal de Minas Gerais. Received for publication August 18, 2014; accepted for publication November 14, 2014. Corresponding Author: Maísa Mota Antunes, MSc, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos 6627, Pampulha, Belo Horizonte 31270-901, Minas Gerais, Brazil. Email: [email protected]
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mucosal inflammation and mucositis.4–6 This agent does not specifically act against cancer cells; it also induces damage to healthy cells that are in active cell division. Thus, the rapidly multiplying cells of normal tissues, such as the gastrointestinal epithelium, are exposed to its action, causing severe impairment of digestive function, including mucositis.7 Mucositis affects 40% of patients undergoing radiotherapy and chemotherapy.8 Therefore, strategies aimed to alleviate symptoms of mucositis are of great benefit. In this regard, the use of immunonutrients has been reported to positively affect the recovery from intestinal lesions, with less side effects.9–11 Arginine and glutamine are specific nutrients whose ability to protect the intestinal mucosa has been previously reported.10,11 Taking into account its lower availability under stressing situations, arginine is considered a conditionally essential amino acid, and higher doses are required to restore plasma levels after challenge.12 Arginine is the major substrate for nitric oxide synthesis, which is involved in many regulatory mechanisms relevant to wound healing, including angiogenesis, cell proliferation, and epithelialization.13 Thus, arginine deficiency will affect organ function and clinical success, causing poor tissue perfusion and microcirculation, as well as increased susceptibility to infections.14–16 However, oral arginine supplementation is inefficient because its excess will be converted within the liver to ornithine and urea, and the increased levels of circulating arginine will upregulate serum levels of arginase, leading to enhanced arginine catabolism.17,18 Although there are no records on human toxicity, administration of higher doses of arginine causes dyspepsia, nausea, and diarrhea.19 Therefore, novel therapeutic venues based on the beneficial effects of arginine might be relevant in the context of cancer management and chemotherapy. L-citrulline, a non-proteinogenic neutral amino acid, has recently been considered a potential nutrient to replace arginine, considering the former’s ability to maintain physiologic serum levels of arginine.18 L-citrulline is produced in the enterocytes, and its advantages over arginine include improved efficacy and increased tissue availability.18,20 Also, L-citrulline is not metabolized by the liver, which leads to reduced urea production and increased arginine synthesis. Moreover, unlike arginine, citrulline transport to the circulation may not be affected during stressing situations.18 In a murine bacterial translocation model, we have previously shown that treatment with L-citrulline preserved intestinal barrier integrity and modulated the immune response, which might explain the reduced bacterial translocation.11 To our knowledge, this is the first study to assess the impact of L-citrulline on the intestinal mucosa in a murine model of 5FU-induced intestinal mucositis.
Materials and Methods Animals and Experimental Design Six-week-old male Swiss mice from the Universidade Federal de Minas Gerais (Brazil) animal facility were fed with commercial
chow and received supplemented citrulline or alanine (ALA) (Sigma-Aldrich, St Louis, MO) in the drinking water for 10 days. Both solutions were isoproteic (1 g/kg/d).21,22 On the seventh day, the animals were injected intraperitoneally with a single dose of phosphate-buffered saline (PBS) or 5FU (200 mg/kg; Faldfluor, lot no. L 11C0041; Libbs, Embu das Artes, São Paulo, Brazil) for the induction of mucositis.23 The animals were randomized into 4 groups: control group (no mucositis and ALA supplementation), CIT group (no mucositis and CIT supplementation), 5FU group (mucositis and ALA supplementation), and 5FU+CIT group (mucositis and CIT supplementation). On day 10, the animals underwent euthanasia, and the blood and small intestines were harvested for the assessment of intestinal histopathology, oxidative stress levels, and myeloperoxidase (MPO) enzyme assay. Another set of animals that received the same treatment was used to assess intestinal permeability. The body weight was measured on the 1st, 7th, and 10th experimental days, and amino acid intake (from supplemented water) and food intake were measured on the 7th and 10th experimental days. The protocol was approved by the Animal Care Committee of the Universidade Federal de Minas Gerais (UFMG; CETEA 231/2011). All experimental procedures were carried out according to the standards set forth in the Guide for the Care and Use of Laboratory Animals of the National Research Council.
Histopathologic Analysis The small intestine, from the pylorus to the ileocecal papilla, was removed. The organ was measured with an inextensible millimeter ruler and gently perfused with paraformaldehyde (3%) for tissue fixation. The intestine was divided into the duodenum, proximal jejunum, distal jejunum, and ileum and then fixed in paraformaldehyde (3%) for 24 hours. The segments were dehydrated, embedded in paraffin, and cut into 5-µm-thick sections before being stained with hematoxylin and eosin (H&E). Digital images were obtained using a 10× objective on a light microscope (Olympus, Center Valley, PA) fitted with a digital camera (Moticam 500, Motic, British Columbia, Canada) for histopathologic evaluation. Alterations of the mucosal architecture (general structure, cell distribution, mucosal and submucosal aspect), the presence of ulcerations, villus height, and the extent of inflammatory infiltration were used to determine the histologic score. The samples were coded and then scored by a trained pathologist who was not aware of the treatment modalities. The score ranged from zero (no alterations) to 3 (severe alterations) (adapted).23,24 The results are presented as the sum of the score obtained for each parameter (0–12).
Oxidative Stress Samples of the small intestine were washed with 1× PBS to remove the intestinal contents. Then, fragments (1 cm) were homogenized with 1 mL cold 1× PBS and centrifuged at 12,000
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rpm for 10 minutes at room temperature. The supernatant was separated for analysis. For evaluation of lipid peroxidation by thiobarbituric acid reactive substances (TBARS), 500 µL of a solution containing trichloroacetic acid (TCA, 15%), thiobarbituric acid (TBA, 0.0375%), and hydrochloric acid (0.25N HCl) was added to the supernatant fragments of duodenum, jejunum, and ileum (250 µL each). The samples were incubated in a boiling water bath for 15 minutes and then placed in water until cool. Then, 750 µL of butyl alcohol was added, and the tubes were vigorously shaken. The samples were centrifuged at 3000 rpm for 10 minutes at room temperature. Next, 200 µL of supernatant was added to a 96-well plate in duplicate. The absorbance was measured spectrophotometrically at 535 nm, and the results were normalized for protein concentration, using the Lowry method.25 The hydroperoxide concentrations were obtained by adding 20 µL of supernatant from each sample directly into the microplate, followed by the FOX-2 reagent. The samples were kept at room temperature for 30 minutes, and the absorbance was measured spectrophotometrically at 560 nm. The second stage of the technique was performed to reduce the hydroperoxides with triphenylphosphine (TPP). First, 5 µL of TPP solution in methanol (10 mM TPP) was added to the supernatants from the duodenum, jejunum, and ileum (15 µL each) directly in the microplate in duplicate. Then, FOX-2 reagent was added, and the absorbance was measured spectrophotometrically at 560 nm. The hydroperoxide quantification was achieved by subtracting the measurements without TPP from those with TPP, and the result was normalized by the protein concentration of each sample.
Enzyme Assay—MPO Activity Neutrophil infiltration was evaluated by analyzing the MPO enzyme activity. The samples were previously washed with 1× PBS to remove the intestinal contents. Then, fragments (1 cm) were homogenized and centrifuged, and the precipitates were used for MPO quantification. Briefly, the precipitates were dissolved in HETAB 0.5% (Sigma-Aldrich) in phosphate buffer and centrifuged. Then, 25 µL of supernatant was added to 25 µL TMB in DMSO (Sigma-Aldrich). After the addition of 100 µL H2O2, the solution was incubated at 37°C for 5 minutes. The reaction was stopped by adding H2SO4, and the plate was read at 450 nm in a microplate spectrophotometer (ThermoPlate, São Paulo, Brazil). The results were expressed in arbitrary units (based on absorbance).
Intestinal Permeability On day 10, animals were gavaged with 0.1 mL of 99mTcdiethyleneaminopentacetic acid (DTPA) labeled with 3.7 MBq 99m Technetium in the form of sodium pertechnetate (Na99mTcO4), obtained by a 99molybdenum/99mtechnetium generator (IPEN/ CNEMA, São Paulo, Brazil). Four hours after the gavage, the
animals were anesthetized and exsanguinated at the axillary plexus. The radioactivity of the standard dose and blood samples was determined in an automatic pit scintillator (ANSR; Abbott Laboratories, Abbott Park, IL), and the percentage of the recovered dose in each animal was calculated as follows: radioactivity of blood/radioactivity of the standard dose 9 × 100.10,26 The average of the control group values was used as the reference. The results were presented as the increase observed in the experimental groups over the control group (expressed as a percentage).
Statistical Analysis Statistical analysis was performed using the GraphPad Prism 5.0 software package (GraphPad Software, San Diego, CA). The results were tested for outliers (Grubbs’ test) and normality using the Kolmogorov-Smirnov test. One-way analysis of variance (ANOVA) and the Newman-Keuls multiple-comparison posttest were used for all parameters except for the architecture alteration and villus height (nonparametric distributions), which were instead analyzed with the Kruskal-Wallis and Dunn posttests. The results were expressed as the mean and standard error or median and interquartile range. A significant difference was defined as P < .05.
Results The amino acid and chow intakes were similar in all groups until the induction of mucositis (days 1–7) (P > .05). However, a reduction in these parameters was observed after the administration of the drug (days 8–10) for the 5FU+CIT group compared with the control group (P < .05), whereas the CIT and 5FU groups presented intermediate intake relative to the control group and the 5FU+CIT group (Table 1).
Weight Change There was no difference in weight between the groups (P > .05) before the induction of mucositis. After the induction of mucositis, weight loss was observed in the 5FU and 5FU+CIT groups, and the loss was greater in the 5FU+CIT group (P < .05) (Figure 1).
Histopathologic Analysis The morphologic structure in the control and CIT groups was within normal limits, with preservation of the villi and epithelium (Figure 2A and 2B). Histologic analyses of the 5FU group showed important tissue damage, reduction in the villus height, and the presence of inflammatory cells and ulcerations all along the small intestine, which was more intense in the proximal jejunum segment (Figure 2C). However, the animals that received L-citrulline presented less tissue damage and partial preservation of the villus length in the
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Table 1. Amino Acid Intake and Chow Intake Before and After the Induction of Mucositis. Group Time, d
Control
Chow intake, g/animal/d 5.580 ± 0.071 1–7 8–10 5.650 ± 0.133a Amino acid intake, g/animal/d 0.067 ± 0.004 1–7 8–10 0.062 ± 0.002a
CIT
5FU
5FU+CIT
5.163 ± 0.171 4.320 ± 0.431a,b
5.493 ± 0.139 4.335 ± 0.673a,b
5.285 ± 0.060 2.860 ± 0.276b
0.052 ± 0.002 0.047 ± 0.004a,b
0.062 ± 0.007 0.050 ± 0.010a,b
0.057 ± 0.004 0.028 ± 0.004b
Results are expressed as the mean ± standard error. Different letters in the same row represent a significant difference (P < .05). n = 4 for all groups. CIT, L-citrulline; 5FU, 5-fluorouracil.
Figure 1. Weight change. Weight change (A) before and (B) after induction of mucositis. Results are expressed as the mean and standard error. Different letters represent a significant difference. One-way analysis of variance and Newman-Keuls multiplecomparison posttest were used (P < .05). CIT, L-citrulline; 5FU, 5-fluorouracil.
proximal jejunum (Figure 2D) but not in the other segments (data not shown). Intestinal injury, as assessed by histopathologic score, was higher in the 5FU group in all of the intestinal segments (Figure 3A–D). Despite the numerically smaller histologic score in the 5FU+CIT group, we could not find any significant difference in comparison to 5FU group (P > .05). However, an intermediate score between the control and the 5FU group was observed in the proximal jejunum for the 5FU+CIT group (Figure 3B).
The CIT and 5FU groups presented with intermediate values of MPO activity (Figure 5).
Oxidative Stress
Discussion
Lipid peroxidation and the concentration of hydroperoxides in the small intestine of all groups and in all intestinal segments were similar (P > .05) (Figure 4).
Chemotherapy is one of the most common treatments in patients with cancer and has a tremendous negative impact on nutrition status, worsening patient condition and enhancing morbidity.27 In particular, 5FU, a drug commonly used to treat malignant tumors such as colorectal cancer, inhibits DNA synthesis, leading to villus and crypt atrophy, which induces severe intestinal toxicity with concomitant mucosal inflammation and mucositis.4–6 We have shown that pretreatment with L-citrulline attenuated intestinal damage in a 5FU-induced murine mucositis model. Interestingly, there is no evidence of off-target effects of L-citrulline in attenuating the chemotherapeutic action of 5FU, supporting its safe use
Neutrophil Infiltration Assessed by MPO Activity Neutrophil infiltration, as assessed by MPO activity, was similar in the duodenum (Figure 5A) and ileum (Figure 5C) (P > .05). However, in the jejunum, the group receiving L-citrulline (5FU+CIT) showed increased neutrophil infiltration in comparison to the control group (Figure 5B) (P < .05).
Intestinal Permeability Intestinal permeability was higher in animals receiving 5FU (P < .05). However, L-citrulline administration reduced intestinal permeability compared with the nonsupplemented group (Figure 6).
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Figure 2. Morphologic structure of the proximal jejunum. Morphology of the proximal jejunum after treatment with phosphatebuffered saline or 5-fluorouracil (5FU). Groups: (A) control, (B) L-citrulline (CIT), (C) 5FU, and (D) 5FU+CIT. The arrow in C points to an area with intense loss of epithelium with inflammatory cells. 10× magnification.
during cancer treatment. In fact, previous studies have used the same model of mucositis associating with other compounds, indicating that no direct effects on the chemotherapeutic effect of 5FU are expected.23,24,28 To our knowledge, this is the first study that has described the role of L-citrulline in gut barrier function in this model. Also, previous studies have assessed the impact of other nutrients such as arginine and glutamine on mucositis in experimental models with controversial outcomes,28,29 and L-citrulline has mostly been used as a biomarker of intestinal failure because it is produced by enterocytes.30 L-citrulline is an important component of the nitric oxide (NO) pathway, and it is a precursor in the arginine cycle. In sharp contrast to arginine, L-citrulline intake is not associated with upregulation of arginase activity, and there is no evidence that the transport of citrulline is impaired in situations of stress.18 Therefore, we suggest that the beneficial effects of L-citrulline supplementation observed, in our hands, might be derived from the provasogenic effects, enhancing tissue repair. Thus, supplementation with CIT may also be efficient in
situations where the availability of arginine is impaired, including patients with mucositis.20 We have observed that the intestinal lesions caused by 5FU impaired nutrient absorption, contributing to higher weight loss. Similar results were reported by Ferreira et al,23 who found, using a similar animal model, greater weight loss and decreased chow intake among the animals that received 5FU relative to the control group. In the present study, mice supplemented with CIT had lower food and water consumption, which may explain the weight loss. However, even with reduced dietary intake and greater weight loss, these mice had a significant improvement of the intestinal mucosa in all evaluated parameters, suggesting that CIT supplementation may have beneficial effects even in lower dosages. 5FU damages intestinal villus architecture, which is observed by decreased villus height, an increased number of inflammatory cells in the lamina propria, and the presence of ulcerations. Our results were similar to Ferreira et al23 and Soares et al,24 who used 200 mg/kg and 150 mg/kg 5FU, respectively. However, mice that received L-citrulline showed
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Figure 3. Histopathologic score of the small bowel. Histopathologic score of the (A) duodenum, (B) proximal jejunum, (C) distal jejunum, and (D) ileum. Results expressed as median and interquartile range. Different letters represent significant differences. Oneway analysis of variance and Kruskal-Wallis and Dunn posttests were used (P < .05). CIT, L-citrulline; 5FU, 5-fluorouracil.
Figure 4. Oxidative stress. Analysis of (A) lipid peroxidation by thiobarbituric acid reactive substances (TBARS) and (B) concentration of hydroperoxide. Results expressed as mean and standard error. Different letters represent significant differences. Oneway analysis of variance and Newman-Keuls multiple-comparison posttest were used (P < .05). CIT, L-citrulline; 5FU, 5-fluorouracil; MDA, malondialdehyde.
less injury compared with the nonsupplemented group, suggesting a protective effect of the amino acid on the intestinal mucosa. Batista et al11 reported similar results in a bacterial translocation murine model in which L-citrulline–treated mice had significantly higher preserved mucosal areas and a larger number of intact villus structures.
The integrity of the intestinal barrier is essential to prevent passage through the luminal epithelium of antigens, toxins, and microorganisms associated with the development of systemic infection and aggravation of existing pathologic conditions.31–33 We observed an increased intestinal permeability in mice with mucositis, which was rescued by supplementation
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Figure 5. Myeloperoxidase (MPO) activity. Assessment of MPO activity in the (A) duodenum, (B) jejunum, and (C) ileum. Results expressed as mean and standard error. Different letters represent significant differences. One-way analysis of variance and NewmanKeuls multiple-comparison posttest were used (P < .05). CIT, L-citrulline; 5FU, 5-fluorouracil.
Figure 6. Intestinal permeability. Intestinal permeability measured by the presence of 99mTc-DTPA in the blood of animals after treatment with 5FU or phosphate-buffered saline. Different letters represent significant differences. One-way analysis of variance and Newman-Keuls multiple-comparison posttest were used (P < .05). CIT, L-citrulline; 5FU, 5-fluorouracil.
with L-citrulline. Batista et al11 had previously demonstrated that treatment with L-citrulline resulted in an improvement of intestinal permeability in a different murine model. Similar results have been observed in intestinal obstruction models involving supplementation with arginine and glutamine.9,10,34
According to Moinard and Cynober,12 L-citrulline may act as an antioxidant free radical scavenger. This feature could help in the maintenance of epithelial integrity, reducing the impact of the injury caused by mucositis. Unexpectedly, we observed no significant alterations in oxidative stress, as assessed by TBARS and hydroperoxide analysis. This might be explained by our shorter duration protocol (euthanasia on day 3 after mucositis induction), which may have precluded the assessment of lipid peroxidation. Inflammatory cells, such as neutrophils and macrophages, generate free radicals responsible for cellular oxidation and subsequent tissue damage, and they reach their peak more rapidly after injury.5 Furthermore, the generation of free radicals and lipid peroxidation reactions, usually measured by the products of malondialdehyde (MDA, the main TBARS), is reached 48 hours after the administration of chemotherapeutic drugs, including methotrexate.35,36 Neutrophils are the first inflammatory cells to arrive at the injured site,37 peaking in cellularity 48 hours after the onset of the lesion. These cells preferentially settle in the lamina propria and the intestinal crypts, and they are responsible for the release of the enzyme MPO and reactive oxygen species, which are crucial to avoid pathogen infiltration and spread during disease. In our study, despite reduced inflammatory signs displayed in the 5FU+CIT group, we found no decrease in neutrophil infiltration in the jejunum, which might explain the preservation of intestinal barrier observed in these mice.
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Conclusions We conclude that in animals undergoing induction chemotherapy with 5FU, pretreatment with L-citrulline reduces the magnitude of damage to the mucosal architecture of the small intestine and decreases intestinal permeability induced by 5FU.
Acknowledgments We thank the students of Laboratório de Aterosclerose e Bioquímica Nutricional (LABiN) for helping in analyses, the students of Laboratório Imunobiofotônica for helping in review, and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Pró-Reitoria de Pesquisa da Universidade Federal de Minas Gerais for financial support.
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16. Popovic PJ, Zeh HJ, Ochoa JB. Arginine and immunity. J Nutr. 2007;137:S1681-S1686. 17. Curis E, Nicolis I, Moinard C, et al. Almost all about citrulline in mammals. Amino Acids. 2005;29:177-205. 18. Romero MJ, Platt DH, Caldwell RB, Caldwell RW. Therapeutic use of citrulline in cardiovascular disease. Cardiovasc Drug Rev. 2006;24: 275-290. 19. Heys SD, Gough DB, Eremin O. Is nutritional support in patients with cancer undergoing surgery beneficial? Eur J Surg Oncol. 1996;22: 292-297. 20. Hickner RC, Tanner CJ, Evans CA, et al. L-citrulline reduces time to exhaustion and insulin response to a graded exercise test. Me Sci Sports Exerc. 2006;38(4):660-666. 21. Osowska S, Moinard C, Neveux N, Loï C, Cynober L. L-citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection. Gut. 2004;53:1781-1786. 22. Osowska S, Neveux N, Nakib S, Lasserre V, Cynober L, Moinard C. Impairment of arginine metabolism in rats after massive intestinal resection: effect of parenteral nutrition supplemented with citrulline compared with arginine. Clin Sci (Lond). 2008;115(5):159-166. 23. Ferreira TM, Leonel AJ, Melo MA, et al. Oral supplementation of butyrate reduces mucositis and intestinal permeability associated with 5-fluorouracil administration. Lipids. 2012;47:669-678. 24. Soares PM, Mota JM, Gomes AS, et al. Gastrointestinal dysmotility in 5-fluorouracil–induced intestinal mucositis outlasts inflammatory process resolution. Cancer Chemother Pharmacol. 2008;63:91-98. 25. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275. 26. Generoso SV, Viana ML, Santos RG, et al. Protection against increased intestinal permeability and bacterial translocation induced by intestinal obstruction in mice treated with viable and heat-killed Saccharomyces boulardii. Eur J Nutr. 2011;50:261-269. 27. Hill A, Kiss N, Hodgson B, Crowe T, Walsh A. Associations between nutritional status, weight loss, radiotherapy treatment toxicity and treatment outcomes in gastrointestinal cancer patients. Clin Nutr. 2001;30: 92-98. 28. Leitão RF, Ribeiro RA, Lira AM, et al. Glutamine and alanyl-glutamine accelerate the recovery from 5-fluorouracil–induced experimental oral mucositis in hamster. Cancer Chemother Pharmacol. 2008;61(2): 215-222. 29. Beutheu S, Ouelaa W, Guérin C, et al. Glutamine supplementation, but not combined glutamine and arginine supplementation, improves gut barrier function during chemotherapy-induced intestinal mucositis in rats. Clin Nutr. 2014;33(4):694-701 30. Vokurka S, Svoboda T, Rajdl D, et al. Serum citrulline levels as a marker of enterocyte function in patients after allogeneic hematopoietic stem cells transplantation—a pilot study. Med Sci Monit. 2013;19:81-85. 31. Sun Z, Wang X, Andersson R. Role of intestinal permeability in monitoring mucosal barrier function. Dig Surg. 1998;15:386-397. 32. Baumgart DC, Dignass AU. Intestinal barrier function. Curr Opin Clin Nutr Metab Care. 2002;5:685-694. 33. Costanza AC, Di Candia AM, Mesquita ET, Villacorta H. O intestino na insuficiência cardíaca: aspectos funcionais, imunológicos e terapêuticos. Rev SOCERJ. 2007;20:443-449. 34. Quirino IEP, Correia MITD, Cardoso VN. The impact of arginine on bacterial translocation in an intestinal obstruction model in rats. Clin Nutr. 2007;26:335-340. 35. Maeda T, Miyazono Y, Ito K, Hamada K, Sekine S, Horie T. Oxidative stress and enhanced paracellular permeability in the small intestine of methotrexate-treated rats. Cancer Chemother Pharmacol. 2010;65:1117-1123. 36. Miyazono Y, Gao F, Hone T. Oxidative stress contributes to methotrexate-induced small intestinaltoxicity in rats. Scand J Gastroenterol. 2004;39:1119-1127. 37. Xavier RJ, Podolsky DK. Unravelling the pathogenesis of inflammatory bowel disease. Nature. 2007;448:427-434.
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research-article2014
PENXXX10.1177/0148607114567508Journal of Parenteral and Enteral NutritionAntunes et al
Brief Communication
Pretreatment With L-Citrulline Positively Affects the Mucosal Architecture and Permeability of the Small Intestine in a Murine Mucositis Model
Journal of Parenteral and Enteral Nutrition Volume XX Number X Month 201X 1–8 © 2015 American Society for Parenteral and Enteral Nutrition DOI: 10.1177/0148607114567508 jpen.sagepub.com hosted at online.sagepub.com
Maísa Mota Antunes, MSc1; Paola Caroline Lacerda Leocádio, MSc1; Lílian Gonçalves Teixeira, PhD2; Alda Jusceline Leonel, PhD3; Denise Carmona Cara, PhD4; Gustavo Batista Menezes, PhD4; Simone de Vasconcelos Generoso, PhD5; Valbert Nascimento Cardoso, PhD6; Jacqueline Isaura Alvarez-Leite, PhD3; and Maria Isabel Toulson Davisson Correia, PhD7
Abstract Background: Mucositis is a common complication in patients undergoing radiotherapy and chemotherapy. It is associated with pain, poor quality of life, and malnutrition, leading to an increased number of hospital admissions and prolonged hospitalization. The use of immunonutrients may be an alternative treatment option, which may help to improve patient outcome. Objective: Here we assessed the impact of L-citrulline (CIT) on a murine model of 5-fluorouracil (5FU)–induced mucositis. Methods: Swiss male mice were randomized into 4 groups: control, CIT, 5FU, and 5FU+CIT. Mice were fed with commercial chow and supplemented with an oral solution of alanine (control and 5FU groups) or CIT (CIT and 5FU+CIT groups). On the seventh day, mice received intraperitoneal phosphatebuffered saline or 5FU (200 mg/kg, single dose) to induce mucositis. On the 10th day, mice were euthanized, and the blood and small intestines were harvested. Body weight, morphology, histopathology score (hematoxylin and eosin) of the small intestine (from 0–12), myeloperoxidase activity, oxidative stress level, and intestinal permeability were assessed. Results: We observed significant weight loss after the administration of 5FU in both treated and control animals. CIT administration contributed to a partial recovery of the mucosal architecture as well as an intermediate reduction of the histopathologic score, and functional intestinal permeability was partially rescued. Conclusions: CIT administration attenuated 5FU-mediated damage to the mucosal architecture of the small intestine, decreasing the size of the injured areas and promoting decreased intestinal permeability. (JPEN J Parenter Enteral Nutr. XXXX;xx:xx-xx)
Keywords L-citrulline; mucositis; 5FU; intestinal permeability
Clinical Relevancy Statement Mucositis affects 40% of patients undergoing chemotherapy and radiotherapy. Pain, nausea, and diarrhea are common symptoms, which can worsen the nutrition status of the patients, with a direct impact on their quality of life. The use of immunonutrients has been reported to positively affect the recovery from intestinal lesions. In this regard, L-citrulline, a non-proteinogenic neutral amino acid, used in this study may be a strategy to alleviate the symptoms of mucositis, with improvement of nutrition status and decreased length of hospital stay.
Introduction Patients with cancer are frequently undernourished, and chemotherapy and radiotherapy increase the risk of mucositis.1 Pain, nausea, vomiting, and diarrhea are common symptoms in these patients, which can worsen their nutrition status and directly affect their quality of life,2 causing longer hospital stays and higher costs.3 Chemotherapy with 5-fluorouracil (5FU), a drug that is still commonly used in oncologic treatment, inhibits DNA synthesis, resulting in increased apoptosis and decreased cell proliferation, leading to villus and crypt atrophy with concomitant
From the 1Departamento de Ciência de Alimentos, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte–MG, Brazil; 2Departamento de Ciência dos Alimentos, Setor de Nutrição, Universidade Federal de Lavras, Lavras–MG, Brazil; 3Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte–MG, Brazil; 4Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte–MG, Brazil; 5Departamento de Nutrição, Escola de Enfermagem, Universidade Federal de Minas Gerais, Belo Horizonte–MG, Brazil; 6Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte–MG, Brazil; and 7Departamento de Cirurgia, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte–MG, Brazil. Financial disclosure: This work was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Pró-Reitoria de Pesquisa da Universidade Federal de Minas Gerais. Received for publication August 18, 2014; accepted for publication November 14, 2014. Corresponding Author: Maísa Mota Antunes, MSc, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos 6627, Pampulha, Belo Horizonte 31270-901, Minas Gerais, Brazil. Email: [email protected]
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mucosal inflammation and mucositis.4–6 This agent does not specifically act against cancer cells; it also induces damage to healthy cells that are in active cell division. Thus, the rapidly multiplying cells of normal tissues, such as the gastrointestinal epithelium, are exposed to its action, causing severe impairment of digestive function, including mucositis.7 Mucositis affects 40% of patients undergoing radiotherapy and chemotherapy.8 Therefore, strategies aimed to alleviate symptoms of mucositis are of great benefit. In this regard, the use of immunonutrients has been reported to positively affect the recovery from intestinal lesions, with less side effects.9–11 Arginine and glutamine are specific nutrients whose ability to protect the intestinal mucosa has been previously reported.10,11 Taking into account its lower availability under stressing situations, arginine is considered a conditionally essential amino acid, and higher doses are required to restore plasma levels after challenge.12 Arginine is the major substrate for nitric oxide synthesis, which is involved in many regulatory mechanisms relevant to wound healing, including angiogenesis, cell proliferation, and epithelialization.13 Thus, arginine deficiency will affect organ function and clinical success, causing poor tissue perfusion and microcirculation, as well as increased susceptibility to infections.14–16 However, oral arginine supplementation is inefficient because its excess will be converted within the liver to ornithine and urea, and the increased levels of circulating arginine will upregulate serum levels of arginase, leading to enhanced arginine catabolism.17,18 Although there are no records on human toxicity, administration of higher doses of arginine causes dyspepsia, nausea, and diarrhea.19 Therefore, novel therapeutic venues based on the beneficial effects of arginine might be relevant in the context of cancer management and chemotherapy. L-citrulline, a non-proteinogenic neutral amino acid, has recently been considered a potential nutrient to replace arginine, considering the former’s ability to maintain physiologic serum levels of arginine.18 L-citrulline is produced in the enterocytes, and its advantages over arginine include improved efficacy and increased tissue availability.18,20 Also, L-citrulline is not metabolized by the liver, which leads to reduced urea production and increased arginine synthesis. Moreover, unlike arginine, citrulline transport to the circulation may not be affected during stressing situations.18 In a murine bacterial translocation model, we have previously shown that treatment with L-citrulline preserved intestinal barrier integrity and modulated the immune response, which might explain the reduced bacterial translocation.11 To our knowledge, this is the first study to assess the impact of L-citrulline on the intestinal mucosa in a murine model of 5FU-induced intestinal mucositis.
Materials and Methods Animals and Experimental Design Six-week-old male Swiss mice from the Universidade Federal de Minas Gerais (Brazil) animal facility were fed with commercial
chow and received supplemented citrulline or alanine (ALA) (Sigma-Aldrich, St Louis, MO) in the drinking water for 10 days. Both solutions were isoproteic (1 g/kg/d).21,22 On the seventh day, the animals were injected intraperitoneally with a single dose of phosphate-buffered saline (PBS) or 5FU (200 mg/kg; Faldfluor, lot no. L 11C0041; Libbs, Embu das Artes, São Paulo, Brazil) for the induction of mucositis.23 The animals were randomized into 4 groups: control group (no mucositis and ALA supplementation), CIT group (no mucositis and CIT supplementation), 5FU group (mucositis and ALA supplementation), and 5FU+CIT group (mucositis and CIT supplementation). On day 10, the animals underwent euthanasia, and the blood and small intestines were harvested for the assessment of intestinal histopathology, oxidative stress levels, and myeloperoxidase (MPO) enzyme assay. Another set of animals that received the same treatment was used to assess intestinal permeability. The body weight was measured on the 1st, 7th, and 10th experimental days, and amino acid intake (from supplemented water) and food intake were measured on the 7th and 10th experimental days. The protocol was approved by the Animal Care Committee of the Universidade Federal de Minas Gerais (UFMG; CETEA 231/2011). All experimental procedures were carried out according to the standards set forth in the Guide for the Care and Use of Laboratory Animals of the National Research Council.
Histopathologic Analysis The small intestine, from the pylorus to the ileocecal papilla, was removed. The organ was measured with an inextensible millimeter ruler and gently perfused with paraformaldehyde (3%) for tissue fixation. The intestine was divided into the duodenum, proximal jejunum, distal jejunum, and ileum and then fixed in paraformaldehyde (3%) for 24 hours. The segments were dehydrated, embedded in paraffin, and cut into 5-µm-thick sections before being stained with hematoxylin and eosin (H&E). Digital images were obtained using a 10× objective on a light microscope (Olympus, Center Valley, PA) fitted with a digital camera (Moticam 500, Motic, British Columbia, Canada) for histopathologic evaluation. Alterations of the mucosal architecture (general structure, cell distribution, mucosal and submucosal aspect), the presence of ulcerations, villus height, and the extent of inflammatory infiltration were used to determine the histologic score. The samples were coded and then scored by a trained pathologist who was not aware of the treatment modalities. The score ranged from zero (no alterations) to 3 (severe alterations) (adapted).23,24 The results are presented as the sum of the score obtained for each parameter (0–12).
Oxidative Stress Samples of the small intestine were washed with 1× PBS to remove the intestinal contents. Then, fragments (1 cm) were homogenized with 1 mL cold 1× PBS and centrifuged at 12,000
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rpm for 10 minutes at room temperature. The supernatant was separated for analysis. For evaluation of lipid peroxidation by thiobarbituric acid reactive substances (TBARS), 500 µL of a solution containing trichloroacetic acid (TCA, 15%), thiobarbituric acid (TBA, 0.0375%), and hydrochloric acid (0.25N HCl) was added to the supernatant fragments of duodenum, jejunum, and ileum (250 µL each). The samples were incubated in a boiling water bath for 15 minutes and then placed in water until cool. Then, 750 µL of butyl alcohol was added, and the tubes were vigorously shaken. The samples were centrifuged at 3000 rpm for 10 minutes at room temperature. Next, 200 µL of supernatant was added to a 96-well plate in duplicate. The absorbance was measured spectrophotometrically at 535 nm, and the results were normalized for protein concentration, using the Lowry method.25 The hydroperoxide concentrations were obtained by adding 20 µL of supernatant from each sample directly into the microplate, followed by the FOX-2 reagent. The samples were kept at room temperature for 30 minutes, and the absorbance was measured spectrophotometrically at 560 nm. The second stage of the technique was performed to reduce the hydroperoxides with triphenylphosphine (TPP). First, 5 µL of TPP solution in methanol (10 mM TPP) was added to the supernatants from the duodenum, jejunum, and ileum (15 µL each) directly in the microplate in duplicate. Then, FOX-2 reagent was added, and the absorbance was measured spectrophotometrically at 560 nm. The hydroperoxide quantification was achieved by subtracting the measurements without TPP from those with TPP, and the result was normalized by the protein concentration of each sample.
Enzyme Assay—MPO Activity Neutrophil infiltration was evaluated by analyzing the MPO enzyme activity. The samples were previously washed with 1× PBS to remove the intestinal contents. Then, fragments (1 cm) were homogenized and centrifuged, and the precipitates were used for MPO quantification. Briefly, the precipitates were dissolved in HETAB 0.5% (Sigma-Aldrich) in phosphate buffer and centrifuged. Then, 25 µL of supernatant was added to 25 µL TMB in DMSO (Sigma-Aldrich). After the addition of 100 µL H2O2, the solution was incubated at 37°C for 5 minutes. The reaction was stopped by adding H2SO4, and the plate was read at 450 nm in a microplate spectrophotometer (ThermoPlate, São Paulo, Brazil). The results were expressed in arbitrary units (based on absorbance).
Intestinal Permeability On day 10, animals were gavaged with 0.1 mL of 99mTcdiethyleneaminopentacetic acid (DTPA) labeled with 3.7 MBq 99m Technetium in the form of sodium pertechnetate (Na99mTcO4), obtained by a 99molybdenum/99mtechnetium generator (IPEN/ CNEMA, São Paulo, Brazil). Four hours after the gavage, the
animals were anesthetized and exsanguinated at the axillary plexus. The radioactivity of the standard dose and blood samples was determined in an automatic pit scintillator (ANSR; Abbott Laboratories, Abbott Park, IL), and the percentage of the recovered dose in each animal was calculated as follows: radioactivity of blood/radioactivity of the standard dose 9 × 100.10,26 The average of the control group values was used as the reference. The results were presented as the increase observed in the experimental groups over the control group (expressed as a percentage).
Statistical Analysis Statistical analysis was performed using the GraphPad Prism 5.0 software package (GraphPad Software, San Diego, CA). The results were tested for outliers (Grubbs’ test) and normality using the Kolmogorov-Smirnov test. One-way analysis of variance (ANOVA) and the Newman-Keuls multiple-comparison posttest were used for all parameters except for the architecture alteration and villus height (nonparametric distributions), which were instead analyzed with the Kruskal-Wallis and Dunn posttests. The results were expressed as the mean and standard error or median and interquartile range. A significant difference was defined as P < .05.
Results The amino acid and chow intakes were similar in all groups until the induction of mucositis (days 1–7) (P > .05). However, a reduction in these parameters was observed after the administration of the drug (days 8–10) for the 5FU+CIT group compared with the control group (P < .05), whereas the CIT and 5FU groups presented intermediate intake relative to the control group and the 5FU+CIT group (Table 1).
Weight Change There was no difference in weight between the groups (P > .05) before the induction of mucositis. After the induction of mucositis, weight loss was observed in the 5FU and 5FU+CIT groups, and the loss was greater in the 5FU+CIT group (P < .05) (Figure 1).
Histopathologic Analysis The morphologic structure in the control and CIT groups was within normal limits, with preservation of the villi and epithelium (Figure 2A and 2B). Histologic analyses of the 5FU group showed important tissue damage, reduction in the villus height, and the presence of inflammatory cells and ulcerations all along the small intestine, which was more intense in the proximal jejunum segment (Figure 2C). However, the animals that received L-citrulline presented less tissue damage and partial preservation of the villus length in the
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Table 1. Amino Acid Intake and Chow Intake Before and After the Induction of Mucositis. Group Time, d
Control
Chow intake, g/animal/d 5.580 ± 0.071 1–7 8–10 5.650 ± 0.133a Amino acid intake, g/animal/d 0.067 ± 0.004 1–7 8–10 0.062 ± 0.002a
CIT
5FU
5FU+CIT
5.163 ± 0.171 4.320 ± 0.431a,b
5.493 ± 0.139 4.335 ± 0.673a,b
5.285 ± 0.060 2.860 ± 0.276b
0.052 ± 0.002 0.047 ± 0.004a,b
0.062 ± 0.007 0.050 ± 0.010a,b
0.057 ± 0.004 0.028 ± 0.004b
Results are expressed as the mean ± standard error. Different letters in the same row represent a significant difference (P < .05). n = 4 for all groups. CIT, L-citrulline; 5FU, 5-fluorouracil.
Figure 1. Weight change. Weight change (A) before and (B) after induction of mucositis. Results are expressed as the mean and standard error. Different letters represent a significant difference. One-way analysis of variance and Newman-Keuls multiplecomparison posttest were used (P < .05). CIT, L-citrulline; 5FU, 5-fluorouracil.
proximal jejunum (Figure 2D) but not in the other segments (data not shown). Intestinal injury, as assessed by histopathologic score, was higher in the 5FU group in all of the intestinal segments (Figure 3A–D). Despite the numerically smaller histologic score in the 5FU+CIT group, we could not find any significant difference in comparison to 5FU group (P > .05). However, an intermediate score between the control and the 5FU group was observed in the proximal jejunum for the 5FU+CIT group (Figure 3B).
The CIT and 5FU groups presented with intermediate values of MPO activity (Figure 5).
Oxidative Stress
Discussion
Lipid peroxidation and the concentration of hydroperoxides in the small intestine of all groups and in all intestinal segments were similar (P > .05) (Figure 4).
Chemotherapy is one of the most common treatments in patients with cancer and has a tremendous negative impact on nutrition status, worsening patient condition and enhancing morbidity.27 In particular, 5FU, a drug commonly used to treat malignant tumors such as colorectal cancer, inhibits DNA synthesis, leading to villus and crypt atrophy, which induces severe intestinal toxicity with concomitant mucosal inflammation and mucositis.4–6 We have shown that pretreatment with L-citrulline attenuated intestinal damage in a 5FU-induced murine mucositis model. Interestingly, there is no evidence of off-target effects of L-citrulline in attenuating the chemotherapeutic action of 5FU, supporting its safe use
Neutrophil Infiltration Assessed by MPO Activity Neutrophil infiltration, as assessed by MPO activity, was similar in the duodenum (Figure 5A) and ileum (Figure 5C) (P > .05). However, in the jejunum, the group receiving L-citrulline (5FU+CIT) showed increased neutrophil infiltration in comparison to the control group (Figure 5B) (P < .05).
Intestinal Permeability Intestinal permeability was higher in animals receiving 5FU (P < .05). However, L-citrulline administration reduced intestinal permeability compared with the nonsupplemented group (Figure 6).
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Figure 2. Morphologic structure of the proximal jejunum. Morphology of the proximal jejunum after treatment with phosphatebuffered saline or 5-fluorouracil (5FU). Groups: (A) control, (B) L-citrulline (CIT), (C) 5FU, and (D) 5FU+CIT. The arrow in C points to an area with intense loss of epithelium with inflammatory cells. 10× magnification.
during cancer treatment. In fact, previous studies have used the same model of mucositis associating with other compounds, indicating that no direct effects on the chemotherapeutic effect of 5FU are expected.23,24,28 To our knowledge, this is the first study that has described the role of L-citrulline in gut barrier function in this model. Also, previous studies have assessed the impact of other nutrients such as arginine and glutamine on mucositis in experimental models with controversial outcomes,28,29 and L-citrulline has mostly been used as a biomarker of intestinal failure because it is produced by enterocytes.30 L-citrulline is an important component of the nitric oxide (NO) pathway, and it is a precursor in the arginine cycle. In sharp contrast to arginine, L-citrulline intake is not associated with upregulation of arginase activity, and there is no evidence that the transport of citrulline is impaired in situations of stress.18 Therefore, we suggest that the beneficial effects of L-citrulline supplementation observed, in our hands, might be derived from the provasogenic effects, enhancing tissue repair. Thus, supplementation with CIT may also be efficient in
situations where the availability of arginine is impaired, including patients with mucositis.20 We have observed that the intestinal lesions caused by 5FU impaired nutrient absorption, contributing to higher weight loss. Similar results were reported by Ferreira et al,23 who found, using a similar animal model, greater weight loss and decreased chow intake among the animals that received 5FU relative to the control group. In the present study, mice supplemented with CIT had lower food and water consumption, which may explain the weight loss. However, even with reduced dietary intake and greater weight loss, these mice had a significant improvement of the intestinal mucosa in all evaluated parameters, suggesting that CIT supplementation may have beneficial effects even in lower dosages. 5FU damages intestinal villus architecture, which is observed by decreased villus height, an increased number of inflammatory cells in the lamina propria, and the presence of ulcerations. Our results were similar to Ferreira et al23 and Soares et al,24 who used 200 mg/kg and 150 mg/kg 5FU, respectively. However, mice that received L-citrulline showed
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Figure 3. Histopathologic score of the small bowel. Histopathologic score of the (A) duodenum, (B) proximal jejunum, (C) distal jejunum, and (D) ileum. Results expressed as median and interquartile range. Different letters represent significant differences. Oneway analysis of variance and Kruskal-Wallis and Dunn posttests were used (P < .05). CIT, L-citrulline; 5FU, 5-fluorouracil.
Figure 4. Oxidative stress. Analysis of (A) lipid peroxidation by thiobarbituric acid reactive substances (TBARS) and (B) concentration of hydroperoxide. Results expressed as mean and standard error. Different letters represent significant differences. Oneway analysis of variance and Newman-Keuls multiple-comparison posttest were used (P < .05). CIT, L-citrulline; 5FU, 5-fluorouracil; MDA, malondialdehyde.
less injury compared with the nonsupplemented group, suggesting a protective effect of the amino acid on the intestinal mucosa. Batista et al11 reported similar results in a bacterial translocation murine model in which L-citrulline–treated mice had significantly higher preserved mucosal areas and a larger number of intact villus structures.
The integrity of the intestinal barrier is essential to prevent passage through the luminal epithelium of antigens, toxins, and microorganisms associated with the development of systemic infection and aggravation of existing pathologic conditions.31–33 We observed an increased intestinal permeability in mice with mucositis, which was rescued by supplementation
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Figure 5. Myeloperoxidase (MPO) activity. Assessment of MPO activity in the (A) duodenum, (B) jejunum, and (C) ileum. Results expressed as mean and standard error. Different letters represent significant differences. One-way analysis of variance and NewmanKeuls multiple-comparison posttest were used (P < .05). CIT, L-citrulline; 5FU, 5-fluorouracil.
Figure 6. Intestinal permeability. Intestinal permeability measured by the presence of 99mTc-DTPA in the blood of animals after treatment with 5FU or phosphate-buffered saline. Different letters represent significant differences. One-way analysis of variance and Newman-Keuls multiple-comparison posttest were used (P < .05). CIT, L-citrulline; 5FU, 5-fluorouracil.
with L-citrulline. Batista et al11 had previously demonstrated that treatment with L-citrulline resulted in an improvement of intestinal permeability in a different murine model. Similar results have been observed in intestinal obstruction models involving supplementation with arginine and glutamine.9,10,34
According to Moinard and Cynober,12 L-citrulline may act as an antioxidant free radical scavenger. This feature could help in the maintenance of epithelial integrity, reducing the impact of the injury caused by mucositis. Unexpectedly, we observed no significant alterations in oxidative stress, as assessed by TBARS and hydroperoxide analysis. This might be explained by our shorter duration protocol (euthanasia on day 3 after mucositis induction), which may have precluded the assessment of lipid peroxidation. Inflammatory cells, such as neutrophils and macrophages, generate free radicals responsible for cellular oxidation and subsequent tissue damage, and they reach their peak more rapidly after injury.5 Furthermore, the generation of free radicals and lipid peroxidation reactions, usually measured by the products of malondialdehyde (MDA, the main TBARS), is reached 48 hours after the administration of chemotherapeutic drugs, including methotrexate.35,36 Neutrophils are the first inflammatory cells to arrive at the injured site,37 peaking in cellularity 48 hours after the onset of the lesion. These cells preferentially settle in the lamina propria and the intestinal crypts, and they are responsible for the release of the enzyme MPO and reactive oxygen species, which are crucial to avoid pathogen infiltration and spread during disease. In our study, despite reduced inflammatory signs displayed in the 5FU+CIT group, we found no decrease in neutrophil infiltration in the jejunum, which might explain the preservation of intestinal barrier observed in these mice.
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Journal of Parenteral and Enteral Nutrition XX(X)
Conclusions We conclude that in animals undergoing induction chemotherapy with 5FU, pretreatment with L-citrulline reduces the magnitude of damage to the mucosal architecture of the small intestine and decreases intestinal permeability induced by 5FU.
Acknowledgments We thank the students of Laboratório de Aterosclerose e Bioquímica Nutricional (LABiN) for helping in analyses, the students of Laboratório Imunobiofotônica for helping in review, and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Pró-Reitoria de Pesquisa da Universidade Federal de Minas Gerais for financial support.
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