Inhibition of Peroxynitrite-Mediated Reactions by Vanillin S. SANTOSH KUMAR, † K. INDIRA PRIYADARSINI, § AND KRISHNA B. SAINIS* ,† Radiation Biology and Health Sciences Division, Bioscience Group, and Radiation Chemistry and Chemical Dynamics Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India Several neurodegenerative diseases such as Alzeimer’s and Parkinson’s as well as septic shock and inflammation involve formation of reactive oxygen and nitrogen species that include peroxynitrite (PON). PON can also react with endogenous antioxidants. Therefore, dietary supplementation with antioxidants may help in these diseases. An exogenous antioxidant, vanillin (4-hydroxy-3-methoxy- benzaldehyde), used widely as a food flavoring agent, was evaluated for its ability to scavenge PON and inhibit PON-mediated reactions. Nitration of tyrosine by PON was assessed by high-performance liquid chromatography (HPLC). This reaction was inhibited by vanillin. The oxidation of dihydro- rhodamine 123 to fluorescent rhodamine 123 was also inhibited by vanillin. The kinetics of reaction between PON and vanillin was studied by stopped-flow technique. The products of this reaction were analyzed by HPLC, and hydroxyvanillin was identified as one of the five products with absorp- tion at 350 nm. These data demonstrate that vanillin effectively scavenges PON in cell-free systems. KEYWORDS: Vanillin; peroxynitrite; phenoxyl radicals; pulse radiolysis; stopped-flow; antioxidant INTRODUCTION Peroxynitrite (PON) is formed in vivo by diffusion-controlled reaction of nitric oxide (NO) with superoxide anions (O 2 •- )(1, 2). Stimulated macrophages, neutrophils, and endothelial cells are known to produce PON, and its in vivo formation has been shown during pathophysiological conditions (3-6). It has been implicated in several neurodegenerative diseases such as Alzhe- imer’s and Parkinson’s, in ischemia-reperfusion injury, and also in septic shock and inflammation (7-10). Under physiological conditions, PON reacts with various cellular macromolecules such as DNA, lipids, and proteins, leading to strand breaks, lipid peroxidation, and nitration of amino acids, respectively (11- 16). In addition, PON reacts with different endogenous anti- oxidants such as ascorbate, R-tocopherol, and thiol-containing molecules, which form the primary defense against oxidative stress, leading to their depletion and reduced availability (17- 20). Hence, exogenous compounds, especially of dietary origin, that are capable of scavenging reactive oxygen/nitrogen species may play a pivotal role in preventing/controlling degenerative diseases. Vanillin, a plant phenol, is used widely as a food flavoring agent in confectioneries, chocolates, butter, toppings, icings, distilled spirits, etc. (21, 22). Its antioxidant activity has been ascertained using diphenylpicrylhydrazyl radical (DPPH) assay in our laboratory (23). Furthermore, it was shown to protect DNA and mitochondrial membrane against oxidative stress in vitro (24-26). Vanillin displayed antimutagenic activity in both bacteria and mammalian cells (27, 28). It reduced chromosomal damage induced by mitomycin C, hydrogen peroxide, and ionizing radiation in cultured Chinese hamster ovary cells (28, 29). It also inhibited chemically induced hepatocarcinogenesis in rat (30, 31). However, evidence for scavenging of highly reactive nitrogen species (RNS) such as PON by vanillin has not been reported so far. The amino acid tyrosine has been shown to be susceptible to the action of PON, and the stable end product, 3-nitrotyrosine, is used as a standard oxidative marker. The ability to inhibit nitrotyrosine formation provides a useful assay to screen compounds for PON scavenging ability (32, 33). The other type of reaction of PON with its substrates is oxidation. The inhibition of PON-induced oxidation of dihydrorhodamine 123 to rhodamine 123 is also a widely used assay for antioxidants (34). The present studies were, therefore, carried out to assess the ability of vanillin to inhibit PON reactions and to separate and identify the products generated during the reaction. The kinetics of the reaction between PON and vanillin was also monitored by different methods. MATERIALS AND METHODS Vanillin, hydroxyvanillin, dihydrorhodamine 123 (DHR123), ty- rosine, and nitrotyrosine were obtained from Sigma-Aldrich Chemical Co., St. Louis, MO. Sodium azide, phosphate salts, potassium nitrite, and potassium nitrate of the highest purity available were obtained locally from Glaxo India Ltd., Mumbai. IOLAR grade gases were used. Synthesis of Peroxynitrite. PON was synthesized by the ozonolysis of alkaline sodium azide solution for 2 h at 0-4 °C as described by * Address correspondence to this author at Associate Director, Bioscience Group, Bhabha Atomic Research Centre, Modular Labs, Trombay, Mumbai 400 085, India (e-mail kbsainis@apsara.barc.ernet.in; fax 91-22-25505326/ 25505151). † Radiation Biology and Health Sciences Division. § Radiation Chemistry and Chemical Dynamics Division. J. Agric. Food Chem. 2004, 52, 139-145 139 10.1021/jf030319d CCC: $27.50 © 2004 American Chemical Society Published on Web 12/09/2003