N-acetyl-L-tryptophan (NAT) provides protection to intestinal epithelial cells (IEC-6) against radiation-induced apoptosis via modulation of oxidative stress and mitochondrial membrane integrity.

Abstract

Ionizing radiation generates oxidative stress in biological systems via inducing free radicals. Gastro-intestinal system has been known for its high radiosensitivity. Therefore, to develop an effective radiation countermeasure for gastrointestinal system, N-acetyl L-tryptophan was evaluated for its radioprotective efficacy using intestinal epithelial cells-6 (IEC-6) cells as the experimental model. Cellular metabolic and lysosomal activity of L-NAT and L-NAT treated irradiated IEC-6 cells were assessed by MTT and NRU staining, respectively. ROS and mitochondrial superoxide levels along with mitochondrial disruption were detected using specific fluorescent probes. Endogenous antioxidants (CAT, SOD, GST, GPx) activities were determined using calorimetric assay. Apoptosis and DNA damage were assessed using flow cytometery and Comet assay, respectively. Results of the study were demonstrated that L-NAT pre-treatment (-1 h) to irradiated IEC-6 cells significantly contribute to ensuring 84.36% to 87.68% (p<0.0001) survival at 0.1 μg/mL concentration against LD50 radiation dose (LD50; 20 Gy). Similar level of radioprotection was observed with a clonogenic assay against γ radiation (LD50; 5 Gy). L-NAT was found to provide radioprotection by neutralizing radiation-induced oxidative stress, enhancing antioxidant enzymes (CAT, SOD, GST, and GPx), and protecting DNA from radiation-induced damage. Further, significant restoration of mitochondrial membrane integrity along with apoptosis inhibition was observed with irradiated IEC-6 cells upon L-NAT pretreatment.