Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with peroxidase activity and Ca 2؉ -independent phospholipase A2 (iPLA2) activity. Here, we report that H 2 O 2 -induced cellular toxicity acts through Prdx6 hyperoxidation. Under high concentrations of H 2 O 2 (>100 M), Prdx6, and 2-Cys Prdxs were hyperoxidized. Contrary to hyperoxidation of 2-Cys Prdxs, hyperoxidation of Prdx6 was irreversible in vivo. Surprisingly, H 2 O 2 -induced cell cycle arrest at the G2/M transition correlated with hyperoxidation and increased iPLA2 activity of Prdx6. This arrest was also associated with up-regulation of p53 and p21 and with downregulation of cyclin B1. Furthermore, the H 2 O 2 -mediated increase in iPLA2 activity was dramatically abolished in a hyperoxidation mutant (C47A), an iPLA2 mutant (S32A), and a double mutant (C47A/S32A) of Prdx6, demonstrating the essential requirement of Prdx6 C47 hyperoxidation for its iPLA2 activity. Together, our results demonstrate that H 2 O 2 -mediated hyperoxidation of Prdx6 induces cell cycle arrest at the G2/M transition through up-regulation of iPLA2 activity. Peroxiredoxins (Prdxs)2 are a family of peroxidases that reduce mainly hydrogen peroxide (H 2 O 2 ) and alkyl hydroperoxides to water and alcohol, respectively (1, 2). Prdxs are classified as either 1-Cys or 2-Cys Prdxs, based on whether the protein contains one or two conserved cysteine residues, respectively. In mammals, six members of the Prdx family have been described. Five of these (Prdx1, Prdx2, Prdx3, Prdx4, and Prdx5) are 2-Cys enzymes that use thioredoxin as the electron donor of their catalytic cycle (3, 4). In contrast, Prdx6, the sole mammalian 1-Cys Prdx, does not use thioredoxin as a reductant.In addition to peroxidase activity, Prdx6 has a Ca 2ϩ -independent phospholipase A2 (iPLA2) activity (5). The peroxidase activity of Prdx6 has been widely studied in cells and animal models for its antioxidant properties that provide protection against the harmful consequence of oxidative stress (6 -8). However, the iPLA2 activity of Prdx6 remains poorly understood. Considering the many functions of iPLA2 activity, including cell cycle progression, apoptosis, and tumorigenesis (9), it may also play an important role in either H 2 O 2 -mediated signaling or H 2 O 2 -related cellular events.Prdxs like Prdx6 use cysteine as a catalytic center, rather than selenocysteine which characterizes the glutathione peroxidases (GPx) (2, 3). The N-terminal conserved cysteines (Cys 51 of Prdx1) of Prdx1-5 are selectively oxidized by H 2 O 2 to Cys-SOH (10). The unstable Cys 51 -SOH reacts with Cys 172-SH of another Prdx molecule, creating a homodimer through an intermolecular disulfide bond. The disulfide is then reduced back to the Prdx active thiol form by the thioredoxin-thioredoxin reductase system (11-16). Under oxidative stress condition, however, the sulfenic intermediate is susceptible to hyperoxidation and thus occasionally hyperoxidized by H 2 O 2 , leading to the formation of sulfinic acid (Cys-SO 2 H) or sulfonic acid (Cys-SO 3...

Oxidative stress is implicated in the pathogenesis of allergic asthma and remains an attractive target for the prevention of the disease. Herein, we investigated the anti-inflammatory effects of apocynin, a NADPH oxidase (NOX) inhibitor, in both in vitro and in vivo allergen-induced experimental asthma mediated by Th2 hyperresponsiveness. Apocynin showed potential antioxidant activities and inhibitory effects on the activation of redox-sensitive transcription factors, such as NF-jB and AP-1, induced by pro-inflammatory stimuli, such as TNF-a, lipopolysaccharide and Poly I:C, and that inhibited the production of pro-inflammatory cytokines, such as TNF-a, IL-1b and IL-6. In in vivo experimental asthma model, moreover, apocynin significantly attenuated ovalbumin-induced airway hyperresponsiveness and inflammation, as shown by the attenuation of total inflammatory cell and soluble product influx into bronchoalveolar lavage fluid, such as macrophages, eosinophils, IL-4, IL-5, IL-12, IL-13 and TNF-a. Apocynin also significantly reduced lung inflammation in the tissues. Altogether, these results suggest that apocynin may be useful in the treatment of inflammatory diseases induced by oxidative stress through NOX activity. Keywords: apocynin; NADPH oxidase; oxidative stress; asthma; inflammation; NF-kB Asthma is a chronic inflammatory lung disease characterized by infiltration of inflammatory cells, including eosinophils, and airway hyperresponsiveness (AHR). 1-5 T-helper type 2 (Th2) cells, together with other inflammatory cells such as macrophages, eosinophils, mast cells and B cells, have critical roles in the initiation, development and chronicity of this disease. 1 Upon challenge with various allergens, these inflammatory cells infiltrate into the airway and contribute to the production of Th2 cytokines, such as IL-4, IL-5 and IL-13, which are found at elevated levels in asthmatic lungs. 1-5 Th2 cytokines are pivotal for B cell maturation, IgR synthesis, airway eosinophilia, mucus secretion and ultimately AHR. Specifically, IL-4 regulates allergic inflammation by promoting Th2 cell differentiation, controlling the production of IgE in B cells, stimulating B cell proliferation, inducing the upregulation of MHC class II molecules and increasing the expression of an inducible form of the low-affinity receptor for IgE (FcRII or CD23) on B lymphocytes and macrophages. 4,5 IL-5 influences the production, maturation and activation of eosinophils. 4,5 IL-13 is a potent modulator of human monocyte and B cell function. 5 IL-13 is also capable of inducing the expression of CD23 on purified human B cells and acts as a switch factor directing IgE synthesis. 5 Increased reactive oxygen species (ROS) generation, which results in imbalance between oxidative forces and the antioxidant defense systems, has been implicated in the pathogenesis of asthma. 6-8 ROS are capable of eliciting a variety of pathological changes, including the peroxidation of lipids, proteins and DNA, and the generation of chemo-attractants, enhancement of AHR,...

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