Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A mucosite induzida por quimioterÃpicos à um efeito colateral importante e limitante da terapia do cÃncer, cuja fisiopatologia nÃo à completamente compreendida. O presente estudo visa investigar o papel do Ãxido nÃtrico (NO) na patogÃnese das mucosites oral e intestinal induzidas por 5-fluorouracil (5-FU) e metotrexato (MTX) e os efeitos da glutamina (GLU) e alanil-glutamina (AL-GLU) na mucosite oral induzida por 5-FU. A mucosite oral foi induzida por duas administraÃÃes intraperitoneais (i.p.) de 5-FU nos 1 e 2 dias (60 e 40 mg/kg respectivamente), em hamsters. Os animais foram tratados subcutaneamente (s.c.) com os inibidores da Ãxido nÃtrico sintase (NOS), N-(3-(Aminomethyl)benzyl)acetamidina (1400W; 1 mg/kg), aminoguanidina (AG; 5 or 10 mg/kg), Nφ-Nitro-L-Arginina Methyl Ester (L-NAME; 5, 10 or 20 mg/kg) ou salina (0,4 ml), uma hora antes do 5-FU e, diariamente, atà o sacrifÃcio, no 10 dia. Em outro ciclo de experimentos, os animais receberam salina, suspensÃo de GLU ou de AL-GLU (100 mM) uma hora antes do 5-FU e, diariamente, atà o sacrifÃcio, nos 10 e 14 dias. A mucosite intestinal foi induzida pela administraÃÃo de MTX (2,5 mg/kg; s.c.) nos primeiros trÃs dias de experimentos, em ratos Wistar. Os animais foram tratados com AG (10 mg/Kg; i.p.), ou L-NAME (20 mg/Kg; i.p.), uma hora antes do MTX e, diariamente, atà o sacrifÃcio, no 5 dia. Na investigaÃÃo do papel do NO na mucosite oral induzida por 5-FU, os seguintes parÃmetros foram avaliados: anÃlises micro e macroscÃpica, atividade de mieloperoxidade (MPO) e da NOS, nÃveis teciduais de nitrito, imunohistoquÃmica para NOSi e detecÃÃo de morte celular. O efeito do 5-FU na produÃÃo salivar tambÃm foi avaliado. No estudo dos efeitos da GLU e AL-GLU, anÃlises micro e macroscÃpicas, atividade de MPO, detecÃÃo de morte celular, estoques teciduais de glutationa e concentraÃÃo sÃrica de glutamina foram os parÃmetros avaliados. No estudo do papel do NO na mucosite intestinal, foram realizados anÃlise histopatolÃgica, medida da altura de vilos nos trÃs segmentos do intestino delgado, atividade de MPO, detecÃÃo de apoptose, assim como, western blot e imunohistoquÃmica para NOSi. 1400W e AG, contrariamente ao L-NAME, reduziram os parÃmetros macro e microscÃpicos da mucosite oral e a infiltraÃÃo de cÃlulas inflamatÃrias, detectada na histopatologia e na atividade de MPO. Foram observados ainda, no 10 dia, maior atividade da NOS e marcaÃÃo imunohistoquÃmica para NOSi. 1400W reverteu a diminuiÃÃo da secreÃÃo salivar induzida por 5-FU. A mucosite oral induzida por 5-FU resultou na diminuiÃÃo dos nÃveis sÃricos de glutamina, bem como dos estoques teciduais de glutationa, no 10 dia, efeitos que foram revertidos pela administraÃÃo de GLU e AL-GLU. Apesar de nÃo ter prevenido a mucosite oral no 10 dia, o tratamento com GLU ou AL-GLU reduziu os parÃmetros macro e microscÃpicos da mucosite oral, e a atividade de MPO, no 14 dia. Na mucosite intestinal, AG e L-NAME preveniram o encurtamento de vilos e reduziram a necrose de criptas, assim como o infiltrado inflamatÃrio, efeitos induzidos pelo MTX, sendo esse Ãltimo constatado pela anÃlise histopatolÃgica e atividade de MPO. Foi detectado maior marcaÃÃo imunohistoquÃmica para NOSi no jejuno de ratos submetidos à mucosite intestinal. Esses resultados sugerem o papel relevante do NO na fisiopatologia das mucosites oral e intestinal. O presente estudo demonstrou ainda que a GLU e AL-GLU aceleraram a recuperaÃÃo da mucosa dos animais submetidos a mucosite oral por 5-FU, aumentando os nÃveis teciduais de glutationa, reduzindo a inflamaÃÃo e promovendo reepitelizaÃÃo / Mucositis induced by antineoplastic drugs is an important, dose-limiting and costly side effect of cancer therapy, which pathophysiology is not completely understood. The aim of the present study was to investigate the role of nitric oxide (NO) on the pathogenesis of oral and intestinal mucositis induced by 5-fluorouracil (5-FU) and methotrexate (MTX) and the effect of glutamine (GLU) and alanyl-glutamine (AL-GLU) on 5-FU-induced experimental mucositis. Oral mucosistis was induced by two intraperitoneal (i.p) administrations of 5-FU on the 1st and 2nd days (60 and 40 mg/kg, respectively) in hamsters. Animals were treated subcutaneously (s.c.) with the nitric oxide synthase (NOS) inhibitors N-(3-(Aminomethyl)benzyl)acetamidine (1400W; 1 mg/kg), aminoguanidine (AG; 5 or 10 mg/kg), Nφ-Nitro-L-Arginine Methyl Ester (L-NAME; 5, 10 or 20 mg/kg) or saline (0.4 ml), one hour before the injections of 5-FU and daily until sacrifice, on the 10th day. In another set of experiments, animals received saline, GLU or AL-GLU suspension (100 mM) one hour before the injections of 5-FU and daily until sacrifice, on the 10th or 14th day. Intestinal mucositis was induced by three administrations of MTX (2.5 mg/kg; s.c.) on the first three days of the experiment, in Wistar rats. Animals were treated i.p. with AG (10 mg/Kg), or L-NAME (20 mg/Kg), one hour before the injections of MTX and daily until sacrifice, on the 5th day. In the investigation of the role of NO on 5-FU induced oral mucositis, the following parameters were evaluated: microscopic and macroscopic analysis, myeloperoxidade (MPO) and NOS activities, nitrite level, immunohistochemistry for NOSi, salivary secretion, and cell death. In order to study the effect of GLU and AL-GLU, microscopic and macroscopic analysis, MPO activity, cell death, glutathione stores and the serum concentration of glutamine, were evaluated. In the MTX-induced intestinal mucositis, histopathological analysis was evaluated and the villus height in all three small intestine segments was measured. MPO activity, cell death, as well as, western blot and immunohistochemistry to evaluated the expression of the NOSi, were also conducted. 1400W or AG, but not L-NAME, reduced macroscopic and histological parameters of oral mucositis, and reduced the inflammatory cell infiltration as detected on histopathology and by MPO activity. Increased NOS activity and immunostaining for NOSi were detected. 5-FU induced a decrease in salivary secrection, observed on 4th day, and this effect was prevented by 1400W. The 5-FU-induced oral mucositis significantly decreased the serum GLU level as well as the cheek pouch glutathione stores, observed on day 10. GLU or AL-GLU reversed the 5-FU effects, restoring serum GLU levels and cheek pouch glutathione stores, observed on day 10, but did not prevent oral mucositis at this time. However, GLU and AL-GLU reduced macroscopic and histological parameters of oral mucositis, and reduced the MPO activity on day 14. In the MTX-induced mucositis, AG and L-NAME significantly prevented villous blunting, lamina propria cell death, and reduced crypt necrosis induced by MTX, decreasing neutrophil infiltration as detected by histopathology and by MPO activity. These data were associated with the detection of iNOS expression by Western blot and by immunohistochemistry in the jejunum tissue. These results suggest an important role of NO in the pathogenesis of oral and intestinal mucositis induced by 5-FU and MTX. The present study also demonstrated that GLU or AL-GLU hastens mucosal recovery increasing mucosal tissue glutathione stores, reducing inflammatory parameters and speeding reepithelization