Abstract
Methotrexate (MTX), a structural analogue of folic acid, is widely used as a chemotherapeutic agent for leukemia and other malignancies. One of the major toxic effects of MTX is intestinal injury and enterocolitis .The mechanism of gastrointestinal toxicity of methotrexate has not been investigated completely. Therefore cancer chemotherapy has to be accompanied by symptomatic therapy such as antibiotics and anti-diarrheal drugs. It is important to investigate the mechanism by which methotrexate induces intestinal damage in order to perform cancer chemotherapy effectively by preventing the side effects. This study aimed at investigating whether nitrosative stress plays a role in methotrexate induced small intestinal damage using a rat model. Adult male rats were administered methotrexate at the dose of 7 mg/kg body weight intraperitoneally for 3 consecutive days and sacrificed 12 or 24 h after the final dose of methotrexate. Vehicle treated rats served as control. The intestinal tissue was used for light microscopic studies and markers of nitrosative stress including tissue nitrite level and nitrotyrosine. Myeloperoxidase (MPO) activity, a marker of neutrophil infiltration was also measured in intestinal homogenates. The villi were damaged at 12 h and the damage progressed and became severe at 24 h after the final dose of MTX. Biochemically, tissue nitrate was elevated fivefold at 12 h and fourfold at 24 h after the final dose of MTX as compared with control. Nitrotyrosine, measured immunohistochemically was detected in all the parts of the small intestine. Duodenum stained the most for nitrotyrosine, followed by ileum and then jejunum. The staining for nitrotyrosine was more intense at 24 h as compared with 12 h after the final dose of methotrexate. There was marked neutrophil infiltration as evidenced by increase in MPO activity in the small intestines. In conclusion, the results of the present study reveal that nitrosative stress may play a critical role in methotrexate induced small intestinal damage. Intervention studies using nitric oxide synthase inhibitors is being carried out in order to confirm the role of nitrosative stress in methotrexate induced small intestinal damage.
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References
Altmann CG (1974) Changes in the mucosa of the small intestine following methotrexate administration or abdominal X-irradiation. Am J Anat 140:263–280
Berezin I, Snyder SH, Bredt DS, Daniel EE (1994) Ultrastructural localization of nitric oxide synthase in canine small intestine and colon. Am J Physiol 266:C981–989
Boughton-Smith NK, Evans SM, Hawkey CJ, Cole AT (1993) Nitric oxide synthase activity in ulcerative colitis and Crohn’s disease. Lancet 342:338–340
Brown JF, Tepperman BL, Hanson PJ (1992) Differential distribution of nitric oxide synthase between cell fractions isolated from the rat gastric mucosa. Biochem Biophys Res Commun 184:680–685
Ciralik H, Bulbuloglu E, Cetinkaya A, Kurutas EB, Celik M, Polat A (2006) Effects of N-acetylcysteine on methotrexate—induced small intestinal damage in rats. Mt Sinai J Med 73:1086–1092
Cunningham D, Morgan RJ, Mills PR (1985) Functional and structural changes of the human proximal small intestine after cytotoxic therapy. J Clin Pathol 38:265–270
Cuzzocrea S, Zingarelli B, Costantino G (1997) Beneficial effects of 3-aminobenzamide, an inhibitor of poly (ADP-ribose) synthetase in a rat model of splanchnic artery occlusion and reperfusion. Br J Pharmacol 121:1065–1074
Diaz-Granados N, Howe K, Lu J, McKay DM (2000) Dextran sulfate sodium-induced colonic histopathology, but not altered epithelial ion transport, is reduced by inhibition of phosphodiesterase activity. Am J Pathol 156:2169–2177
Dickinson E, Tuncer R, Nadler E (1999) NOX, a novel nitric oxide scavenger, reduces bacterial translocation in rats after endotoxin challenge. Am J Physiol 277:G1281
Doan T, Massarotti E (2005) Rheumatoid arthritis: an overview of new and emerging therapies. J Clin Pharmacol 45:751–762
Estervez AG, Radi R, Barbeito L (1995) Peroxynitrite-induced cytotoxicity in PC 12 cells: evidence for an apoptotic mechanism differentially modified by neutrotrophic factors. J Neurochem 65:1543–1549
Ford H, Watkins S, Reblock K, Rowe M (1997) The role of inflammatory cytokines and nitric oxide in the pathogenesis of necrotizing enterocolitis. J Pediatr Surg 32:275–282
Hackam DJ, Upperman JS, Grishin A, Ford HR (2005) Disodered enterocyte signaling and intestinal barrier dysfunction in the pathogenesis of necrotizing enterocolitis. Semin Pediatr Surg 14:49–57
Helen STE, Schiano TD, Kuan SF, Hanauer SB, Conjeevaram HS (2000) Hepatic effects of long-term methotrexate use in the treatment of inflammatory bowel disease. Am J Gastroenterol 95:3150–3156
Hoffman RA, Zhang G, Nussler NC (1997) Constitutive expression of inducible nitric oxide synthase in the mouse ileal mucosa. Am J Physiol 272:G383–G392
Ignarro LJ (1990) Biosynthesis and metabolism of endothelium-derived nitric oxide. Annu Rev Pharmacol Toxicol 30:535–560
Jolivet J, Cowan KH, Curt GA, Clendeninn NJ, Chabner BA (1983) The pharmacology and clinical use of methotrexate. N Engl J Med 309:1094–1104
Kavuklu B, Agalar C, Guc MO (1998) Evidence that aminoguanidine inhibits endotoxin induced bacterial translocation. Br J Surg 85:1103
Kehoe JE, Harvey LP, Daly JM (1986) Alteration of chemotherapy toxicity using a chemically defined liquid diet in rats. Cancer Res 46:4047–4052
McAnena OJ, Rossi M, Mehta BM, Daly JM (1987) Alteration of methotrexate metabolism in rats by administration of an elemental liquid diet I. Changes in drug enterohepatic circulation. Cancer 59:31–37
Middleton SJ, Shorthouse M, Hunter JO, Cole AT (1993) Increased nitric oxide synthesis in ulcerative colitis. Lancet 341:465–466
Nadler EP, Upperman JS, Dickinson EC, Ford HR (1999) Nitric oxide and intestinal barrier failure. Semin Pediatr Surg 8:148–154
Nagakubo J, Tomimatsu T, Kitajima M, Takayama H, Aimi N, Horie T (2001) Characteristics of transport of fluoresceinated methotrexate in rat small intestine. Life Sci 69:739–747
Phelen MJ, Taylor W, van Heyningen C, Williams E, Thompson RN (1993) Intestinal absorption in patients with rheumatoid arthritis treated with methotrexate. Clin Rheumatol 12:223–225
Potoka DA, Nadler EP, Upperman JS, Ford HR (2002) Role of nitric oxide and peroxynitrite in gut barrier failure. World J Surg 26:806–811
Radi R (2004) Nitric oxide, oxidants, and protein tyrosine nitration. Proc Natl Acad Sci USA 101:4003–4008
Radi R, Beckman JS, Bush KM (1991) Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch Biochem Biophys 288:481
Razzouk BI, Rose SR, Hongeng S, Wallace D, Smeltzer MP, Zacher M, Pui CH, Hudson MM (2007) Obesity in survivors of childhood acute lymphoblastic leukemia and lymphoma. J Clin Oncol 25:1183–1189
Sastry KV, Moudgal RP, Mohan J, Tyagi JS, Rao GS (2002) Spectrophotometric determination of serum nitrite and nitrate by copper–cadmium alloy. Anal Biochem 306:79–82
Sener G, Eksioglu-Demiralp E, Cetinar M, Ercan F, Yegen BC (2006) β glucan ameliorates methotrexate-induced oxidative organ injury via its antioxidant and immunomodulatory effects. Eur J Pharmacol 542:170–178
Singer II, Kawka DW, Scott S (1996) Expression of inducible nitric oxide synthase and nitrotyrosine in colonic epithelium in inflammatory bowel disease. Gastroenterology 111:871–885
Sorrells DL, Friend C, Koltuksuz U, Courcoulas A (1996) Inhibition of nitric oxide with aminoguanidine reduces bacterial translocation after endotoxin challenge in vivo. Arch Surg 131:1155–1163
Spies M, Chappel VL, Dasu MR, Herdon DN (2002) Role of TNF α in gut mucosal changes after severe burn. Am J Physiol Gastrointest Liver Physiol 283:G703–G708
Sullivan GW, Sarembock IJ, Linden J (2000) The role of inflammation in vascular diseases. J Leukoc Biol 67:591–602
Tepperman BL, Brown JF, Whittle BJR (1993) Nitric oxide synthase induction and intestinal cell viability in rats. Am J Physiol 265:G214–218
Unno N, Wang H (1997) Inhibition of inducible nitric oxide synthase ameliorates endotoxin induced gut barrier dysfunction in rats. Gastroenterology 113:1246
van’t Land B, Meiijer HP, Frerich J (2002) Transforming growth factor beta 2 protects the small intestine during methotrexate treatment in rats possibly by reducing stem cell cycling. Br J Cancer 87:113–118
Verburg M, Renes IB, Einerhand AW, Buller HA, Dekker J (2003) Isolation-stress increases small intestinal sensitivity to chemotherapy in rats. Gastroenterology 124:660–671
Wallace JL, Macnaughton W K, Morris GP, Beck PL (1989) Inhibition of leukotriene synthesis markedly accelerates healing in a rat model of inflammatory bowel disease. Gastroenterology 96:29–36
Warden RA, Noltorp RS, Lynn Francis J, Dunkley PR, O’Loughlin EV (1997) Vitamin A deficiency exacerbates methotrexate-induced jejunal injury in rats. J Nutr 127:770–776
Yanez JA, Teng XW, Roupe KA, Fariss MW, Davies NM (2003) Chemotherapy induced gastrointestinal toxicity in rats: involvement of mitochondrial DNA, gastrointestinal permeability and cyclooxygenase-2. J Pharm Pharm Sci 6:308–311
Acknowledgments
The authors acknowledge the Council for Scientific and Industrial Research (CSIR), New Delhi for the financial support for the study. Mr. Viswa Kalyan Kolli is a junior research fellow under the present study. Dr Bina Isaac, Professor in Anatomy is acknowledged for her assessment in light microscopic studies.
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Kolli, V.K., Abraham, P. & Rabi, S. Methotrexate-induced nitrosative stress may play a critical role in small intestinal damage in the rat. Arch Toxicol 82, 763–770 (2008). https://doi.org/10.1007/s00204-008-0287-9
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DOI: https://doi.org/10.1007/s00204-008-0287-9