Enzyme-Mediated Protein Haptenation of Dapsone and Sulfamethoxazole in Human Keratinocytes: II. Expression and Role of Flavin-Containing Monooxygenases and Peroxidases

Abstract

Arylamine compounds, such as sulfamethoxazole (SMX) and dapsone (DDS), are metabolized in epidermal keratinocytes to arylhydroxylamine metabolites that auto-oxidize to arylnitroso derivatives, which in turn bind to cellular proteins and can act as antigens/immunogens. Previous studies have demonstrated that neither cytochromes P450 nor cyclooxygenases mediate this bioactivation in normal human epidermal keratinocytes (NHEKs). In this investigation, we demonstrated that methimazole (MMZ), a prototypical substrate of the flavin-containing monooxygenases (FMOs), attenuated the protein haptenation observed in NHEKs exposed to SMX or DDS. In addition, recombinant FMO1 and FMO3 were able to bioactivate both SMX and DDS, resulting in covalent adduct formation. Western blot analysis confirmed the presence of FMO3 in NHEKs, whereas FMO1 was not detectable. In addition to MMZ, 4-aminobenzoic acid hydrazide (ABH) also attenuated SMX-and DDS-dependent protein haptenation in NHEKs. ABH did not alter the bioactivation of these drugs by recombinant FMO3, suggesting its inhibitory effect in NHEKs was due to its known ability to inhibit peroxidases. Studies confirmed the presence of peroxidase activity in NHEKs; however, immunoblot analysis and reverse transcription-polymerase chain reaction indicated that myeloperoxidase, lactoperoxidase, and thyroid peroxidase were absent. Thus, our results suggest an important role for FMO3 and yet-to-be identified peroxidases in the bioactivation of sulfonamides in NHEKs.

Biotransformation of Drugs in Human Skin

“…Although lactoperoxidase (Gorlewska-Roberts et al, 2004), myeloperoxidase (Uetrecht et al, 1993), and thyroid peroxidase (Gupta et al, 1992) have been shown to bioactivate arylamines to reactive metabolites in vitro, none of these enzymes seems to be expressed in cultured human KCs (Vyas et al, 2006b). However, it is noteworthy that peroxidase inhibition reduces the bioactivation of dapsone in human KC cultures (Vyas et al, 2006b)-substantiating a role for peroxidases in cutaneous bioactivation. Further studies are needed to identify the specific peroxidases expressed in human skin and skin cells.…”

“…The N-hydroxylation of dapsone and sulfamethoxazole in cultures of KCs is somewhat surprising in view of the absence of CYP2C9 expression in these cells (Reilly et al, 2000). Although CYP2C9 is the enzyme responsible for the N-hydroxylation of these drugs in liver, it seems that FMO3 and peroxidases are responsible for this metabolism in KCs (Vyas et al, 2006b). Because the arylhydroxylamine metabolites of these drugs are believe to be involved in the provocation of an immune response (Cribb et al, 1996), this observation suggests that local biotransformation may be important in these reactions in the skin.…”

“…Expression of the flavin monooxygenase (FMO) family of enzymes has been probed by two groups. Whereas FMO1 mRNA was detected in skin biopsy samples, neither message nor protein were detected in cultures of KCs or HaCaT cells (Janmohamed et al, 2001;Vyas et al, 2006b). In contrast, FMO3 message was expressed in skin biopsy samples, KCs, and HaCaT cells.…”

Dual Oxidase 1 Induced by Th2 Cytokines Promotes STAT6 Phosphorylation via Oxidative Inactivation of Protein Tyrosine Phosphatase 1B in Human Epidermal Keratinocytes

“…Although DUOX1 is known to play a role in supplying H 2 O 2 to thyroperoxidase and lactoperoxidase in thyroid and nonthyroid tissues, respectively (34), it was unlikely that DUOX1 plays either of these roles in NHEK, because Vyas et al (35) have clearly demonstrated the lack of thyroperoxidase or lactoperoxidase in NHEK.…”

Formation and Uptake of Arylhydroxylamine-Haptenated Proteins in Human Dendritic Cells

“…Because peroxidases and FMO have been shown to bioactivate DDS and SMX in human epidermal keratinocytes (Vyas et al, 2006b), leading to protein haptenation, we sought to determine the role of these enzymes in the DDS-specific protein haptenation in naive KG-1 cells. For this purpose, naive KG-1 cells were exposed to DDS in the presence or absence of a peroxidase inhibitor (ABH) or a competitive substrate for FMO (MMZ).…”