Reactive oxygen species generation and its role in the differential cytotoxicity of the arylhydroxylamine metabolites of sulfamethoxazole and dapsone in normal human epidermal keratinocytes Piyush M. Vyas a,b , Sanjoy Roychowdhury a , Patrick M. Woster b , Craig K. Svensson a,b, * a Division of Pharmaceutics, College of Pharmacy, The University of Iowa, 115 S. Grand Avenue, S213 PHAR, Iowa City, IA 52242, USA b Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI, USA Received 19 February 2005; accepted 20 April 2005 Abstract Cutaneous drug reactions (CDR) are responsible for numerous minor to life-threatening complications. Though the exact mechanism for CDR is not completely understood, evidence suggests that bioactivation of drugs to reactive oxygen or nitrogen species is an important factor in the initiation of these reactions. Several CDR-inducing drugs having an arylamine functional group, such as sulfamethoxazole (SMX) and dapsone (DDS), undergo bioactivation to reactive arylhydroxylamine metabolites. These metabolites can generate cellular oxidative stress by forming reactive oxygen species (ROS). Several studies have demonstrated a higher cytotoxicity with DDS hydroxylamine (DDS-NOH) compared to SMX hydroxylamine (SMX-NOH). To investigate the role of differential ROS generation in the higher cytotoxicity of DDS-NOH, hydroxylamine metabolites of SMX and DDS were synthesized and ROS formation by these metabolites determined. DDS-NOH and its analogues/metabolites consistently resulted in higher ROS formation as compared to SMX- NOH. However, comparison of the ROS generation and cytotoxicity of a series of arylhydroxylamine analogues of DDS did not support a simple correlation between ROS generation and cell death. Numerous ROS scavengers were found to reduce metabolite-induced ROS formation, with differences in the potency between the agents. The decrease in DDS-NOH-induced ROS generation in NHEK with ascorbic acid, N-acetylcysteine, Trolox, and melatonin was 87, 86, 44, and 16%, respectively. Similarly, the cytotoxicity and adduct formation of DDS-NOH in NHEK was reduced in the presence of ascorbic acid. In summary, these studies show that arylhydroxylamine metabolites of SMX/DDS induce ROS generation in NHEK, though such generation is not directly related to cytotoxicity. # 2005 Elsevier Inc. All rights reserved. 1. Introduction Sulfamethoxazole (SMX) is an effective therapeutic agent in the treatment of Pneumocystis carinii pneumonia (PCP), resulting in the recovery of approximately 75% of patients suffering from this ailment [1,2]. Dapsone (DDS) is a useful alternative treatment for PCP, used either alone or in combination with other agents, in patients that cannot tolerate SMX [3–5]. Introduction of the use of these agents for the treatment of PCP in patients infected with the human immunodeficiency virus (HIV) was associated with a high level of adverse reactions, including delayed-type hypersensitivity reactions [6–10]. Numerous studies have suggested that these idiosyncratic reactions result from the bioactivation of these agents to arylhydroxylamine metabolites [11–13]. The in vitro cytotoxicity of arylhydroxylamine metabo- lites of SMX (SMX-NOH) and DDS (DDS-NOH) towards peripheral blood mononuclear cells (PBMCs) has been proposed as a marker for delayed-type hypersensitivity reactions associated with these drugs [14–16]. Although SMX-NOH and DDS-NOH both cause concentration- dependent increases in cell death, DDS-NOH is signifi- cantly more toxic [17]. The in vitro cytotoxicity in human PBMCs exhibits an LC 50 of 325 mM for DDS-NOH and 1752 mM for SMX-NOH. Studies in normal human epidermal keratinocytes (NHEK) demonstrate an LC 50 of 293 mM for DDS-NOH and LC 50 of greater than 1500 mM for SMX-NOH [18]. We have recently observed similar differences in the cytotoxicity of these two arylhydroxy- lamines in normal human dermal fibroblasts (unpublished observations). Differential toxicity is also observed with methemoglobin formation in vitro, with an EC 50 of 26.5 mM for DDS-NOH and of 463 mM for SMX-NOH in human red blood cells [19]. www.elsevier.com/locate/biochempharm Biochemical Pharmacology 70 (2005) 275–286 * Corresponding author. Tel.: +1 319 335 8823; fax: +1 319 335 9349. E-mail address: craig-svensson@uiowa.edu (C.K. Svensson). 0006-2952/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2005.04.023