Characterization of monochloramine toxicity on PC12 cells and protective effect of tocopherol via antioxidative function Rosaria Piga, Yoshiro Saito * , Zhihua Chen, Yasukazu Yoshida, Etsuo Niki Human Stress Signal Research Center (HSSRC), National Institute of Advanced Industrial Science and Technology (AIST), Ikeda, Osaka 563-8577, Japan Received 11 November 2004, and in revised form 17 January 2005 Abstract Monochloramine (NH 2 Cl) is a physiological oxidant produced by activated neutrophils. In the present work, we studied the underlying mechanism of cytotoxic effects of NH 2 Cl on an undifferentiated rat pheochromocytoma PC12 cell line and the protective effects of antioxidants. The cells treated with 100 lM NH 2 Cl exhibited signs of apoptotic cell death such as phosphatidylserine exposure and caspase activation. To understand the mechanism of NH 2 Cl cytotoxicity, we examined the effect of various kinds of antioxidants including a-tocopherol (a-Toc) and b-tocopherol (b-Toc). These antioxidants exerted a protective effect against NH 2 Cl-induced cell death, and a-Toc exhibited the most potent inhibitory effect among the antioxidants used. A loss of cellular glutathione was observed in the cells treated with 100 lM NH 2 Cl. The formation of reactive oxygen species (ROS) was also mea- sured using the fluorescent probe dichlorofluorescin diacetate. The fluorescence intensity increased prior to cell death and an anti- oxidant, such as a-Toc, suppressed the increase in ROS. Interestingly, b-Toc also exerted similar inhibitory effects on cytotoxicity and caspase activation. These results suggest that free radical mediated process is involved in NH 2 Cl-induced PC12 cell death and that tocopherols inhibit this cell death via antioxidative function. Ó 2005 Elsevier Inc. All rights reserved. Keywords: a-Tocopherol; b-Tocopherol; Apoptosis; Monochloramine; Reactive oxygen species Neutrophils are the earliest inflammatory cell to infil- trate tissue and dominate the acute inflammatory stage, playing an important role in the development of inflam- mation site. Activated neutrophils participate in numer- ous tissue injuries including nervous systems [1]. Any lesion induced by infection, ischemia, or trauma in the ner- vous system is followed by an inflammatory process. This process induces rapid activation of glial cells and addi- tional recruitment of neutrophils, T-cells, and monocytes from the blood stream [2]. Monochloramine (NH 2 Cl) 1 is a physiological oxidant derived from activated neutro- phils and plays an important role in tissue injuries [3– 6]. It can be formed by the reaction of hypochlorous acid (HOCl) with NH þ 4 that is spontaneously produced from cell metabolism, taurine, or other amines [7]. Monochloramine is a stable oxidizing agent that readily penetrates the membranes of target cells and thus oxi- dizes the membranes [8]. It has been reported that chlor- amines, including NH 2 Cl, are reactive toward thiol and thioethers [3,9] and react with intracellular components. Furthermore, a variety of biological effects have been reported, including inhibition of protein kinase C activa- tion [10], induction of calcium influx [11], cell cycle ar- rest [12], and apoptosis [13]. The role of radicals derived from chloramines on injury of low density lipo- protein (LDL) and plasma protein has been demon- strated [14–16]. The protective effect of ascorbate and 0003-9861/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2005.01.021 * Corresponding author. Fax: +81 72 751 9964. E-mail address: yoshiro-saito@aist.go.jp (Y. Saito). 1 Abbreviations used: a-Toc, a-tocopherol; DCFH-DA, 2 0 ,7 0 -dichlo- rofluorescin diacetate; GSH, glutathione; MTT, 3-(4,5-dimethylthia- zol-2-yl)-2,5-diphenyl tetrazolium bromide; NAC, N-acetylcysteine; NH 2 Cl, monochloramine; PBS, phosphate-buffered saline; PC12, rat pheochromocytoma cell line; ROS, reactive oxygen species. Archives of Biochemistry and Biophysics 436 (2005) 101–109 www.elsevier.com/locate/yabbi ABB