A Role for Bioactivation and Covalent Binding within Epidermal Keratinocytes in Sulfonamide-Induced Cutaneous Drug Reactions

Abstract

Cutaneous reactions are the most common manifestation of delayed-type hypersensitivity caused by sulfamethoxazole and dapsone. In light of the recognized metabolic and immunologic activity of the skin, we investigated the potential role of normal human epidermal keratinocytes in the development of these reactions. Adult and neonatal normal human epidermal keratinocytes metabolized sulfamethoxazole and dapsone to N-4-hydroxylamine and N-acetyl derivatives in a time-dependent manner. The latter was catalyzed by N-acetyltransferase 1 alone as normal human epidermal keratinocytes did not express mRNA for N-acetyltransferase 2. Investigation of metabolism-dependent toxicity of sulfamethoxazole and dapsone, and subsequent incubation of normal human epidermal keratinocytes with the respective hydroxylamine metabolites, demonstrated that these cells were resistant to the cytotoxic effects of sulfamethoxazole hydroxylamine but not dapsone hydroxylamine. With prior depletion of glutathione, however, normal human epidermal keratinocytes became susceptible to the toxicity of sulfamethoxazole hydroxylamine. Covalent adduct formation by sulfamethoxazole hydroxylamine was detected in normal human epidermal keratinocytes, even in the absence of cell death, and was increased with glutathione depletion. Major protein targets of sulfamethoxazole hydroxylamine were observed in the region of 160, 125, 95, and 57 kDa. Dapsone hydroxylamine also caused covalent adduct formation in normal human epidermal keratinocytes. Together, these observations provide a basis for our hypothesis that normal human epidermal keratinocytes are involved in the initiation and propagation of a cutaneous hypersensitivity response to these drugs.

Bioactivation, protein haptenation, and toxicity of sulfamethoxazole and dapsone in normal human dermal fibroblasts☆

“…DDS and SMX were obtained from Sigma-Aldrich (St. Louis, MO) and found to be >99% pure. Rabbit antisera were raised against SMX-and DDS-keyhole limpet hemocyanine (KLH) conjugates and assayed for specificity as described previously (Reilly et al, 2000). Goat anti-rabbit antibody conjugated with alkaline phosphatase, goat-anti-rabbit IgG conjugated with Alexafluor 488, Yo-Yo-1 and monochlorobimane were purchased from Molecular Probes (Eugene, OR).…”

“…The cell suspension was centrifuged at 220 × g for 5 min and the pellet containing cell debris discarded. The supernatant containing cellular soluble proteins was collected for protein assay and subsequent ELISA analysis of covalent adducts as described previously (Reilly et al, 2000), with minor modifications.…”

“…Biotransformation of SMX and DDS to their arylhydroxylamine (S-NOH or D-NOH) or Nacetyl metabolites in NHDF were determined using methods similar to those described previously (Reilly et al, 2000), with minor modifications. N-hydroxy and N-acetyl metabolites of DDS and SMX were quantified based upon externally generated standard curves using HPLC methods described previously (Reilly et al, 2000).…”

“…In particular, the bioactivation of sulfonamides within keratinocytes to their respective arylhydroxylamine metabolites may result in protein haptenation. Indeed, we have found that incubation of normal adult human epidermal keratinocytes (NHEK) with the arylhydroxylamine metabolites of sulfamethoxazole (SMX) or dapsone (DDS) results in the formation of drug/metabolite-protein adducts (Reilly et al, 2000). In addition, we have recently demonstrated that incubation of these cells with the parent compounds also gives rise to protein haptenation ; supporting a potential role for intracellular bioactivation.…”

“…We have previously proposed that events occurring at the cutaneous level are critical in the initiation of CDRs to sulfonamides and related drugs (Reilly et al, 2000). In particular, the bioactivation of sulfonamides within keratinocytes to their respective arylhydroxylamine metabolites may result in protein haptenation.…”