NF-κB-iNOS-COX2-TNF α inflammatory signaling pathway plays an important role in methotrexate induced small intestinal injury in rats.

Although methotrexate is widely used in clinics as an anticancer agent, it's utility is limited by its gastrointestinal toxicity, the mechanism of which is unclear. The role of NFκB inflammatory pathway in MTX induced mucositis was investigated in the present study. GI injury was induced in adult Wistar rats by the administration of 3 consecutive i.p . injections of MTX. On the fourth day, the rats were sacrificed and the small intestine was removed; A piece was used for light microscopy, immunohistochemistry, immunofluorescence studies . The mucosa was collected and used for the analysis of protein and mRNA expressions of NFκB and its target genes by the western blot, RT-PCR respectively. MTX treatment resulted in NFκB activation and nuclear translocation as evidenced by immunofluorescence, immunohistochemistry , and western blot. NFκB mRNA was also increased. There was increased protein and mRNA expressions of NFκB target genes, TNF-α, iNOS, COX-2, PLA2, HO-1, HSP70, MMPs 2 and 9 . Aminoguanidine pretreatment (30mg/ 50mg /kg body wt.) attenuated MTX induced activation of NFκB and its proinflammatory target genes and improved MTX induced morphological changes. Aminoguanidine has protective effects against MTX induced gastrointestinal mucositis in rats.

Activation of the mitochondrial apoptotic pathway contributes to methotrexate-induced small intestinal injury in rats.

The efficacy of methotrexate (MTX), a commonly used chemotherapeutic drug, is limited by intestinal injury. As the mechanism of MTX-induced small intestinal injury is not clear, there is no definitive treatment for MTX-induced gastrointestinal injury. The present study investigates the role of mitochondrial apoptotic pathway in MTX-induced small intestinal injury and examines whether aminoguanidine is effective in preventing the damage. Eight Wistar rats were administered 3 consecutive i.p. injections of 7 mg/kg body wt. MTX. Some rats were pretreated with 30 mg or 50 mg/kg body wt. of aminoguanidine (n = 6 in each group). Protein expressions of cytochrome c, caspases 3 and 9, and PARP-1 were determined in the small intestines by immunohistochemistry and western blot. Mitochondrial pathway of apoptosis was activated in the small intestines of MTX-treated rats as evidenced by intense immunostaining for cyt c, caspases 9 and 3, and PARP-1 and mitochondrial release of cyt c, activation of caspases, and PARP-1 cleavage by Western blot. Immunofluorescence revealed increased nuclear localization of PARP-1. Aminoguanidine pretreatment ameliorated MTX-induced small intestinal injury in dose-dependent manner and inactivated the mitochondrial apoptotic pathway. Aminoguanidine may possess beneficial intestinal protective effects as an adjuvant co-drug against MTX intestinal toxicity during cancer chemotherapy. As the mechanism of MTX-induced small intestinal injury is not clear, there is no definitive treatment for MTX-induced gastrointestinal injury. The results of the present study show that the mitochondrial pathway of apoptosis plays a role in MTX-induced small intestinal injury as evidenced by cytochrome c release, activation of caspases 9 and 3, PARP-1 cleavage, and DNA fragmentation. Aminoguanidine (AG) pretreatment attenuates the severity of small-intestinal injury induced in rats by MTX treatment. The mechanisms of action of AG involve inhibition of iNOS, and mitochondrial pathway of apoptosis. It is suggested that aminoguanidine may possess beneficial intestinal protective effects as an adjuvant co-drug against MTX intestinal toxicity during cancer chemotherapy.

Generation of an induced pluripotent stem cell line that mimics the disease phenotypes from a patient with Fanconi anemia by conditional complementation.

Generation of Fanconi anemia (FA) patient-specific induced pluripotent stem cells (iPSCs) has been reported to be technically challenging due to the defects in the FA-pathway in the patients' somatic cells. By inducible complementation of FA-pathway, we successfully reprogrammed the fibroblasts of an FA patient to iPSCs. CSCR19i-indCFANCA, one of the iPSC lines generated by the inducible complementation of FA-pathway, was extensively characterized for its pluripotency and karyotype. In the absence of doxycycline (DOX) and FANCA expression, this line showed the cellular phenotypes of FA, suggesting it is an excellent tool for FA disease modeling and drug screening.

Aminoguanidine pretreatment prevents methotrexate-induced small intestinal injury in the rat by attenuating nitrosative stress and restoring the activities of vital mitochondrial enzymes.

BACKGROUND: One of the major toxic side effects of methotrexate (MTX) is enterocolitis, for which there is no efficient standard treatment. Nitric oxide overproduction has been reported to play an important role in MTX-induced mucositis. This study was designed to investigate whether pretreatment with aminoguanidine (AG) - a selective iNOS inhibitor - prevents MTX-induced mucositis in rats. METHODS: Rats were pretreated with AG (30 and 50 mg/kg body weight) i.p. daily 1 h before MTX (7 mg/kg body weight) administration for 3 consecutive days. After the final dose of MTX, the rats were killed, and the small intestines were used for analysis. RESULTS: The small intestines of MTX-treated rats showed moderate to severe injury. Pretreatment with AG had a dose-dependent protective effect on MTX-induced mucositis. AG pretreatment reduced iNOS protein levels, mucosal nitric oxide levels, and protein tyrosine nitration. AG pretreatment also restored the activities of electron transport chain (ETC) complexes, vital tricarboxylic acid (TCA cycle) enzymes, and mitochondrial antioxidant enzymes. CONCLUSIONS: These findings suggest that AG is beneficial in ameliorating MTX-induced enteritis in rats.

Methotrexate administration induces differential and selective protein tyrosine nitration and cysteine nitrosylation in the subcellular organelles of the small intestinal mucosa of rats.

Gastrointestinal toxicity is one of the most frequent dose limiting side effects of methotrexate (MTX), a commonly used chemotherapeutic drug. Peroxynitrite (PON) overproduction is reported to contribute to MTX induced gastrointestinal mucositis. However, the consequence of PON overproduction i.e. protein tyrosine nitration and protein cysteine nitrosylation, the subcellular distribution of these modified proteins and their molecular weights have not been investigated yet. Mucositis was induced in Wistar rats by the administration of 3 consecutive i.p. injections of MTX. Tyrosine nitrated proteins and cysteine nitrosylated proteins were determined in the subcellular organelles fractions of mucosa using immunoprecipitation and western blot. The proteins in the subcellular fractions were separated by 1D electrophoresis, and probed with anti -nitrotyrosine antibody and anti-nitrosocysteine antibody. After MTX treatment, a general increase in protein tyrosine nitration as well as a change in the spectrum of proteins that underwent nitration was observed. The relative densities of the 3 nitrotyrosine protein adducts were as follows: Mitochondria > cytosol > microsomes > nucleus. In the mitochondrial fraction increased nitration of 12 kDa, 25 kDa 29Kda, 47 kDa, and 62Kda proteins, in the cytosol increased nitration of 12 kDa, 19 kDa, 45 kDa, and 60 kDa proteins and in the nuclear fraction increased nitration of 17 kDa, 35 kDa, and 58 kDa proteins was observed. On the other hand, MTX treatment resulted to a general decrease in protein cysteine nitrosylation in all the subcellular fractions. These results suggest that MTX induced, PON mediated small intestinal injury is mediated by differential nitration and nitrosylation of proteins in the subcellular organelles with increased protein tyrosine nitration and decreased cysteine nitrosylation. In addition MTX treatment results in selective nitration and nitrosylation of proteins in the intestinal mucosa. This differential nitrosative modifications may contribute to MTX induced small intestinal injury.

A preclinical study on the protective effect of melatonin against methotrexate-induced small intestinal damage: effect mediated by attenuation of nitrosative stress, protein tyrosine nitration, and PARP activation.

PURPOSE: One of the major toxic side effects of methotrexate (MTX) is enterocolitis. To date, there is no efficient standard treatment for this side effect. Nitrosative stress is reported to play a critical role in MTX-induced mucositis. The purpose of this study is to investigate whether pretreatment with melatonin, an inhibitor of nitro-oxidative stress, prevents MTX-induced mucositis in rats. METHODS: Rats were pretreated with melatonin (20 and 40 mg/kg body weight) i.p. daily 1 h before MTX (7 mg/kg body weight) administration for three consecutive days. After the final dose of MTX, the rats were killed and the small intestines were used for analysis. RESULTS: The small intestines of MTX-treated rats showed moderate to severe injury. The villi were distorted, blunted, and atrophied and focally absent in various segments of the small intestines. Crypt abscesses were also found, suggesting an inflammatory response. Pretreatment with melatonin had a dose-dependent protective effect on MTX-induced mucositis. Morphology was saved to a moderate extent with 20 mg melatonin pretreatment, and near-normal morphology was achieved with 40 mg melatonin pretreatment. Damage to the villi and crypt abscess was reduced. The villi/crypt ratio was almost restored. Melatonin pretreatment protected the small intestines from MTX-induced damage by attenuating nitrosative stress, protein tyrosine nitration and PARP expression. CONCLUSION: Because of its versatility in protecting against nitro-oxidative stress and reducing inflammation, we suggest that melatonin could be beneficial in ameliorating MTX-induced enteritis in humans.

Oral glutamine attenuates cyclophosphamide-induced oxidative stress in the bladder but does not prevent hemorrhagic cystitis in rats.

Cyclophosphamide (CP) is widely used in the treatment of cancer and non-malignant disease states such as rheumatoid arthritis. Hemorrhagic cystitis is a major dose-limiting side effect of CP. The incidence of this side effect is related to the dosage and can be as high as 75%. Elimination of the side effects of CP can lead to better tolerance of the drug, and a more efficient therapy can be achieved for patients in need of CP treatment. Several studies have demonstrated that oxidative stress and neutrophil infiltration play important roles in CP-induced bladder damage. Glutamine is utilized under clinical conditions for preventing chemotherapeutic drug-induced side effects, based on its ability to attenuate oxidative stress. The aim of the study is to verify whether glutamine prevents CP-induced oxidative stress and bladder damage using a rat model. Adult male rats were administered 150 mg/kg body weight of CP intraperitoneally. Glutamine pretreated rats were administered 1 g/kg body weight of glutamine orally 2 h before the administration of CP. Vehicle/glutamine-treated rats served as controls. All the rats were killed 16 h after the dose of CP/vehicle. The urinary bladders were removed and used for light microscopic and biochemical studies. The markers of oxidative stress including malondialdehyde content, protein carbonyl content, protein thiol, and myeloperoxidase activity, a marker of neutrophil infiltration, were measured in bladder homogenates. CP treatment induced hemorrhagic cystitis in the rats. Pretreatment with glutamine significantly reduced CP-induced lipid peroxidation (p < 0.01), protein oxidation (p < 0.01), and increase in myeloperoxidase activity (p < 0.05). However, it did not prevent CP-induced bladder damage. The results of the present study show that glutamine pretreatment does not attenuate CP-induced hemorrhagic cystitis, although it prevents CP-induced oxidative stress and neutrophil infiltration significantly. It is therefore necessary to clarify the utility of glutamine as a chemoprotective agent before it is recommended in the market as a nutrient supplement.