Evaluation of the in vivo antioxidative activity of redox nanoparticles by using a developing chicken egg as an alternative animal model

Antioxidants have been demonstrated to exert beneficial effects as pharmacotherapies for cardiovascular diseases. The in vitro systems generally employed to evaluate antioxidants, however, are limited by having no appreciable in vivo redox status of the antioxidants. Therefore, we used our developing chicken egg model to evaluate the in vivo antioxidative activity of a redox nanoparticle possessing 2,2,6,6-tetramethylpiperidine1- oxyl (RNPO). The 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) elicited strong oxidative stress and its LD50 value for chick embryos was 3.5 +/- 0.9 mg/egg. The lowmolecular weight nitroxide compound, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), which is known to have the highest level of antioxidant activity, showed no significant protective effect against AAPH-induced embryo lethality. On the contrary, RNPO had potent protective effects against AAPH-induced embryo lethality. Moreover, RNPO could significantly suppress the production of lipid peroxides in chick serum induced by hydrocortisone. Since RNPO has a longer retention time in blood than TEMPOL, RNPO may protect the embryo against lethal oxidative stress by suppressing lipid peroxidation. The validity of in vivo experiments using developing chicken eggs was supported by our data, where RNP(O)was determined to elicit strong antioxidative activity in vivo, irrespective of the lack of a significant difference in the in vitro activity between low-molecularweight TEMPOL and RNPO. Our results support the use of the developing chicken egg model to evaluate the potential in vivo antioxidative activity of RNPO. (C) 2014 Elsevier B.V. All rights reserved.