Original Contribution Antioxidant-mediated augmentation of ozone-induced membrane oxidation $ Carol A. Ballinger a , Rafael Cueto b , Giuseppe Squadrito a , Jennifer F. Coffin a , Leonard W. Velsor c , William A. Pryor b , Edward M. Postlethwait a, * a Department of Environmental Health Sciences, School of Public Health, RBPH 530, 1530 3rd Avenue South, University of Alabama at Birmingham, Birmingham, AL 35294-0022, USA b Biodynamics Institute, Louisiana State University, Baton Rouge, LA, USA c Department of Medicine, National Jewish Hospital, Denver, CO, USA Received 6 August 2004; revised 3 November 2004; accepted 5 November 2004 Available online 28 November 2004 Abstract The pulmonary epithelial lining fluid (ELF) contains substrates, e.g., ascorbic acid (AH 2 ), uric acid (UA), glutathione (GSH), proteins, and unsaturated lipids, which undergo facile reaction with inhaled ozone (O 3 ). Reactions near the ELF gas/liquid interface likely provide the driving force for O 3 absorption (breactive absorptionQ) and constrain O 3 diffusion to the underlying epithelium. To investigate the potential mechanisms wherein O 3 /ELF interactions may induce cellular damage, we utilized a red cell membrane (RCM) model intermittently covered by an aqueous film to mimic the lung surface compartmentation, and evaluated exposure-mediated loss of acetylcholinesterase activity (AChE) and TBARS accumulation. In the absence of aqueous reactants, O 3 exposure induced no detectable changes in AChE or TBARS. AH 2 and GSH preferentially induced oxidative damage in a dose-dependent fashion. AH 2 -mediated RCM oxidation was not inhibited by superoxide dismutase, catalase, mannitol, or Fe chelators. O 3 reaction with UA, Trolox, or albumin produced no RCM oxidation but oxidation occurred when AH 2 was combined with UA or albumin. Rat bronchoalveolar lavage fluid (BALF) also induced RCM oxidation. However, in vivo O 3 exposure dampened the extent of BALF-mediated RCM oxidation. Although we cannot completely rule out O 3 diffusion to the RCM, product(s) derived from O 3 + AH 2 /GSH reactions (possibly O 3 S À or 1 O 2 ) likely initiated RCM oxidation and may suggest that in vivo, such secondary species account for O 3 permeation through the ELF leading to cellular perturbations. D 2004 Elsevier Inc. All rights reserved. Keywords: Ozone; Antioxidants; Ascorbic acid; Glutathione; Uric acid; Epithelial lining fluid; Lung; Membrane oxidation; Acetylcholinesterase; Lactate dehydrogenase; Ozonide radical; Singlet oxygen; Free radicals Introduction Ozone (O 3 ) is a ubiquitous toxicant due to its presence in photochemical smog. Environmental ozone exposures are associated with numerous respiratory symptoms including decreased lung function, exacerbation of asthma, airway injury, and inflammation [1–3]. Upon exposure, epithelial injury begins to occur rapidly [4,5], suggesting that during the acute phases of exposure, cellular necrosis predominates due to direct cytotoxicity. Although the precise mechanisms by which O 3 initiates acute lung injury remain equivocal, physical contact between inhaled O 3 and the airspace epithelia may be limited [6–8]. The lung surface is overlain 0891-5849/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2004.11.009 Abbreviations: AChE, acetylcholinesterase; AH 2 , ascorbic acid; AO, ascorbate oxidase; BALF, bronchoalveolar lavage fluid; BSA, bovine serum albumin; CAT, catalase; DFX, desferrioxamine; DTNB, dithiobisni- trobenzoic acid; DTPA, diethylenetriaminepentaacectic acid; ELF, epithelial lining fluid; GSH, reduced glutathione; GSSG, glutathione disulfide; LDH, lactate dehydrogenase; MAN, mannitol; NEM, N-ethylmaleimide; O 3 , ozone; ozonide radical, O 3 S À ; PB, 310 mOsm phosphate buffer, pH 7.4; PO 4 , 10 mOsm phosphate buffer, pH 7.0; RCM, red cell membranes; ROS, reactive oxygen species; 1 O 2 , singlet oxygen; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances; UFA, unsaturated fatty acids; UA, uric acid. $ This work was supported by NIH Grant HL 054696 (E.M.P.). * Corresponding author. Fax: (205) 975 6371. E-mail address: epostlethwait@ms.soph.uab.edu (E.M. Postlethwait). Free Radical Biology & Medicine 38 (2005) 515 – 526 www.elsevier.com/locate/freeradbiomed