The data obtained in our work showed that AMD was

The data obtained in our work showed that AMD was able to inhibit complex I activity and ATP biosynthesis in MRC-5 cells. These effects were accompanied by an increase in lipid and protein oxidative damage and a decrease in NO levels and superoxide dismutase and catalase activities, suggesting that AMD toxicity was related to O2-· and H2O2. Respiratory chain complex I is the most structurally and functionally complex respiratory enzyme [29,30]. Complex I dysfunction increases O2-· production, which can be a substrate for superoxide dismutase originating H2O2, which, in turn, can be a substrate for catalase. O2-· and the H2O2 can also decrease complex I activity [31], which can feed a vicious cycle of complex I inhibition and maintain a state of cellular oxidative stress. Among the factors able to trigger the intrinsic apoptotic pathway are the oxidative stress, depolarization of the mitochondrial inner membrane, and increased release of cytochrome c [32]. Therefore, the mitochondrial dysfunction and redox imbalance induced by AMD could explain, at least, in part, MRC-5 cell death, and might be related to the lung toxicity caused by this antiarrhythmic drug. It is important to mention that these effects could be due to AMD itself and its metabolite N-desethylamiodarone. In further studies, it would be interesting to examine the effects of AMD metabolite to better understand its mechanism of action. Both phenolic compounds CAT and EPI were able to prevent both complex I inhibition and decrease in ATP biosynthesis induced by AMD in MRC-5 cells. Consequently, the formation of O2-· and H2O2, oxidative damage, and death of MRC-5 apigenin were reduced. In addition, CAT and EPI minimized the reduction in NO levels induced by AMD in MRC-5 cells (Fig. 5). These results were similar for both CAT and EPI, which suggests that the chemical difference of the compounds (Fig. 2) was not related to the biological effects demonstrated by CAT and EPI. A study evaluating the ability of CAT and EPI to scavenge the O2-· and reduce the radical 2,2-diphenyl-1-picrylhydrazyl in vitro[33], also did not observe a difference in the effect of the two phenolic compounds. Additional studies using different classes of phenolic compounds would contribute to a better understanding of the relationship between the structure and biological activity of these compounds. The mechanism by which CAT and EPI modulate the activity of complex I is not yet fully known. However, studies have already shown that CAT, resveratrol, and quercetin [34] are capable of directly or indirectly increasing proteins called sirtuins. These classes of molecules are mainly protein deacetylases involved in diverse cellular process and pathways, and they vary in cell localization and functions. Seven sirtuins have already been described in mammals, named SIRT1 to SIRT7. SIRT1 predominately localizes in the nucleus and regulates mitochondrial processes, stress response, cell proliferation, and apoptosis [35]. Furthermore, SIRT1 was found to be associated with vasodilation in rat aortic endothelial cells by increasing the activity of nitric oxide synthase [36]. SIRT3 is the major mitochondrial deacetylase, and it regulates the complex I activity, maintaining electron chain function, and therefore, ATP biosynthesis [35]. However, from the data obtained in our study, it is not possible to determine whether CAT and EPI maintain complex I activity and ATP biosynthesis, thus improving MRC-5 cell viability by directly or indirectly targeting these sirtuins. It has already been shown that EPI-rich cocoa increased the expression of SIRT1 and SIRT3 in skeletal muscle of patients with type II diabetes and heart failure [26]. Other studies should be conducted to clarify this observation and to provide perspectives for the use of sirtuins as new targets to treat AMD toxicity.
Funding This research was supported by a grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; Grant number 302885/2011-0). Mirian Salvador is the recipient of a CNPq Research Fellowship.