Title

 

Authors

P.C. Braga, R. Calò, L. Marabini
Dept. of Pharmacology, School of Medicine, University of Milan, Milan, Italy 

 

Abstract

Erdosteine was originally developed as a mucomodulatory (mucolytic) agent for the treatment of respiratory diseases. The molecule is a pro-drug because, although it does not have a free SH group itself, it produces an active metabolite (MET I) containing a pharmacologically active SH group after first-pass metabolism. It has more recently been shown that in vitro Met I inhibits oxidative stress mediators (ROS, RNS, peroxynitrite) during the respiratory burst of human neutrophils, and also inhibits elastase release. MET I has free radical scavenging and anti-inflammatory activities, which could be widely exploited in COPD  and other lung diseases such as pulmonary fibrosis in which oxidative stress plays an important pathogenetic role.Oxidative stress is a major mechanism for cell damage followed by a variety of secondary cellular processes. DNA is one of the most sensitive biomolecules to the action of ROS and free radicals, which may rupture single or double strands and this damage the bases or break hydrogen bonds.The aim of this study was to investigate the protective effect of MET I against H₂O₂-induced oxidative stress and oxidative damage DNA using a comet assay of A549  human lung epithelial cells, obtained from the American Type Cell Culture Collection and maintained in RPMI-1640. Cell viability was assessed using an MTT assay (Foldbjerg et al.,2010). The generation of intracellular reactive oxygen species was measured using a 2’,7’-dichlorofluorescin diacetate (DCFH-DA) probe (Foldbjerg et al.,2010). The single-cell gel electrophoresis (SCGE) COMET assayis a very sensitive method for detecting DNA alkali-labile sites and strand breaks in individual cells, and it was carried out according to the protocols of Singh et al.,1988, and Gminski et al.,2010.The data were statistically analysed using GraphPad Prism 6.0 (GraphPad Software, Inc. California, USA), and the X² test for the comet assay (evaluation of DNA damage using a visual scoring system based on arbitrary classification) and one-way ANOVA (Bonferroni’s post hoc test) for the intracellular ROS measurements and comet assay (tail moment and % tail DNA).When A549 cells were pre-treated with the active metabolite I (2.5-20µg/ml) for 10-30 min and then exposed to H₂O₂  (1-4 mM) for two additional hours at 37°C, 5% at CO₂, the intracellular peroxide production, reflected by dichlorofluorescein (DCF) fluorescence, decreased in a statistically significant concentration–dependent manner. Furthermore, using a comet assay as an indicator for oxidative DNA damage, we found that the MET I significantly prevented damage to cells exposed to short-term H₂O₂ treatment. Our data suggest that MET I is effective in preventing H₂O₂-induced oxidative stress and DNA damage in A549 cells. The underlying mechanisms involve the scavenging of intracellular reactive oxygen species (ROS).
 
Foldbjerg R. et al. (2010). Arch Toxicol.DOI: 10.1007/s00204-010-0545.
Singh NPet al. (1988). Exp Cell Res.175, 184-191.
Gminski Ret al. (2010). Environ Mol Mutagen.  DOI: 10.1002/em.