Aim: Methotrexate (MTX) is a structural analogue of folic acid and is widely used in the treatment of leukaemia and other malignancies, rheumatoid arthritis and other rheumatic disorders. Gastrointestinal mucositis is a frequent dose-limiting side effect of methotrexate (MTX) chemotherapy. The underlying mechanisms of the mucositis is not completely understood. In the present study we examined the effect of MTX treatment on iNOS gene expression, protein tyrosine nitration protein cysteine nitrosylation, and the activities of mitochondrial enzymes in the small intestinal mucosa of rats

Methods: Gastrointestinal injury was induced in the rats by the administration of 3 consecutive i.p. injections of 7 mg /kg body wt. MTX .The small intestines were used for light microscopy and immunohistochemical localisation of iNOS, nitrotyrosine (NTy) and nitrocysteine (NCy). Mucosal scrapings were used for NOS mRNA expression by RTPCR, iNOS protein, nitrotyrosine and protein cysteine adducts formation by western blot and assay of key mitochondrial enzymes. Results: MTX treatment resulted in moderate to severe damage to the small intestines. iNOS mRNA expression , iNOS protein, NTy protein adduct and NCy adducts were increased in the MTX treated rat intestines. The activities of electron transport chain (ETC) complexes IV and V were decreased by 66% and 71 % respectively in the MTX treated rat intestines. With respect to tricarboxylic acid (TCA) cycle enzymes, aconitase activity was decreased by 73 %, succinate dehydrogenase by 85 % and glutamate dehydrogenase by 61%. With respect to antioxidant enzymes, superoxide dismutase (SOD) activity was increased by 40 %, and that of catalase, glutathione reductase, were decreased by 68 % and 66%, respectively. Conclusion: The gastrointestinal toxicity induced by MTX may be due to increased nitrosative stress. The decreased activities of the mitochondrial energy producing enzymes and antioxidant enzyme may be due to their inactivation by MTX induced nitric oxide overproduction