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CELLULAR AND MOLECULAR

Characterization of the Formation and Localization of Sulfamethoxazole and Dapsone-Associated Drug-Protein Adducts in Human Epidermal Keratinocytes

Division of Pharmaceutics, College of Pharmacy, University of Iowa, Iowa City, Iowa (S.R., P.M.V., C.K.S.); Immunotoxicology, Drug Safety Evaluation, Pharmaceutical Research Institute, Bristol-Myers-Squibb Company, Syracuse, New York (T.P.R.); and Department of Dermatology, University of Maryland School of Medicine, Baltimore, Maryland (A.A.G.)

Sulfonamide- and sulfone-induced hypersensitivity reactions are thought to be mediated through bioactivation of parent drug molecule(s) to their respective reactive metabolite(s). Recent studies have demonstrated that keratinocytes can bioactivate sulfonamides and sulfones. Using enzyme-linked immunosorbent assay and hapten-specific rabbit antisera developed in our laboratory, we found that incubation of either normal human epidermal keratinocytes (NHEKs) or an immortalized human keratinocyte cell line (HaCaT) with sulfamethoxazole (SMX) or dapsone (DDS) resulted in the formation of drug/metabolite protein adducts. The formation of these adducts with SMX was increased in the presence of ascorbic acid, whereas N-acetylcysteine decreased adduct formation with both SMX and DDS. Adduct formation was confirmed using confocal microscopy when NHEKs were incubated with SMX, DDS, or their respective arylhydroxylamine metabolites. Cellular distribution of adducts was compared in permeable versus nonpermeable NHEKs. Exposure to SMX, DDS, or dapsone hydroxylamine resulted in the formation of intracellular adducts, whereas SMX hydroxylamine also resulted in the presence of adducts on the cell surface. In summary, our work shows that keratinocytes can bioactivate SMX/DDS to form drug-protein adducts, which may be acquired by antigen-presenting cells upon keratinocyte cell death, evoking an immune response. In addition, keratinocytes may themselves present antigen to hapten-specific cytotoxic T lymphocytes. Furthermore, our results also suggest that different sulfonamides/sulfones may have different protein targets for in situ haptenation in keratinocytes.

Address correspondence to: Dr. Craig K. Svensson, Division of Pharmaceutics, College of Pharmacy, The University of Iowa, 115 South Grand Avenue, S213 PHAR, Iowa City, IA 52242. E-mail: craig-svensson{at}uiowa.edu

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