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.
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- "Neither CYPs nor cyclooxygenases mediate this bioactivation of sulfamethoxazole and dapsone in NHECs. Methimazole, a prototypical substrate of FMOs, inhibited the bioactivation of sulfamethoxazole and dapsone in the NHECs (Vyas et al. 2006). Diphenylthiourea, a catalytic enhancer used during neoprene synthesis, was bioactivated by FMO-catalyzed S-oxygenation (Samuelsson et al. 2011). "
ABSTRACT: The exposure of the skin to medical drugs, skin care products, cosmetics, and other chemicals renders information on xenobiotic-metabolizing enzymes (XME) in the skin highly interesting. Since the use of freshly excised human skin for experimental investigations meets with ethical and practical limitations, information on XME in models comes in the focus including non-human mammalian species and in vitro skin models. This review attempts to summarize the information available in the open scientific literature on XME in the skin of human, rat, mouse, guinea pig, and pig as well as human primary skin cells, human cell lines, and reconstructed human skin models. The most salient outcome is that much more research on cutaneous XME is needed for solid metabolism-dependent efficacy and safety predictions, and the cutaneous metabolism comparisons have to be viewed with caution. Keeping this fully in mind at least with respect to some cutaneous XME, some models may tentatively be considered to approximate reasonable closeness to human skin. For dermal absorption and for skin irritation among many contributing XME, esterase activity is of special importance, which in pig skin, some human cell lines, and reconstructed skin models appears reasonably close to human skin. With respect to genotoxicity and sensitization, activating XME are not yet judgeable, but reactive metabolite-reducing XME in primary human keratinocytes and several reconstructed human skin models appear reasonably close to human skin. For a more detailed delineation and discussion of the severe limitations see the "Overview and Conclusions" section in the end of this review.Archive für Toxikologie 11/2014; 88(12). DOI:10.1007/s00204-014-1382-8 · 5.08 Impact Factor
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- "Contradictory results were obtained for the distribution of the dermal FMO isoform (Hu et al., 2010; Jackh et al., 2011). Vyas et al. (2006b) and Hu et al. (2010) did not identify FMO in the EpiDerm skin model and in HaCaT cells. "
ABSTRACT: Abstract In general, xenobiotic metabolizing enzymes (XMEs) are expressed in lower levels in the extrahepatic tissues than in the liver, making the former less relevant for the clearance of xenobiotics. Local metabolism, however, may lead to tissue-specific adverse responses, e.g. organ toxicities, allergies or cancer. This review summarizes the knowledge on the expression of phase I and phase II XMEs and transporters in extrahepatic tissues at the body's internal-external interfaces. In the lung, CYPs of families 1, 2, 3 and 4 and epoxide hydrolases are important phase I enzymes, while conjugation is less relevant. In skin, phase I-related enzymatic reactions are considered less relevant. Predominant skin XMEs are phase II enzymes, whereby glucuronosyltransferases (UGT) 1, glutathione-S-transferase (GST) and N-acetyltransferase (NAT) 1 are important for detoxification. The intestinal epithelium expresses many transporters and phase I XME with high levels of CYP3A4 and CYP3A5 and phase II metabolism is mainly related to UGT, NAT and Sulfotransferases (SULT). In the kidney, conjugation reactions and transporters play a major role for excretion processes. In the bladder, CYPs are relevant and among the phase II enzymes, NAT1 is involved in the activation of bladder carcinogens. Expression of XMEs is regulated by several mechanisms (nuclear receptors, epigenetic mechanisms, microRNAs). However, the understanding why XMEs are differently expressed in the various tissues is fragmentary. In contrast to the liver - where for most XMEs lower expression is demonstrated in early life - the XME ontogeny in the extrahepatic tissues remains to be investigated.Drug Metabolism Reviews 03/2014; 46(3). DOI:10.3109/03602532.2014.900565 · 6.29 Impact Factor
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- "FMO3 transcription was restricted to the dermis (Wiegand et al., 2008; Jaeckh et al., 2011). In another study, FMO1 transcription was absent in primary keratinocytes (Vyas et al., 2006b), the keratinocyte-based EpiDerm skin model, and in HaCaT cells (Hu et al., 2010). FMO1 and -3 enzyme activities were assessed in microsomes of abdominal skin biopsies, PFT, EFT, EpiDerm, and KeratinoSens using the model substrate benzydamine. "
ABSTRACT: Skin exposure to sensitizing chemicals can induce allergic reactions. Certain chemicals, so called pro-sensitizers, need metabolic activation to become allergenic. Their metabolic activation occurs in skin cells such as keratinocytes or dendritic cells. These cell types are also incorporated into dermal in vitro test systems used to assess the sensitizing potential of chemicals for humans. In vitrosystems range from single cell cultures to organotypic multi-cellular reconstructed skin models. Until now, their metabolic competence to unmask sensitizing potential of pro-sensitizers was rarely investigated. This review aims to summarize current information on available skin in vitro models and the relevance of xenobiotic metabolizing enzymes for the activation of pro-sensitizers such as eugenol, 4-allylanisole, and ethylendiamine. Among others, these chemicals are discussed as performance standards to validate new coming in vitro systems for their potential to identify pro-sensitizers.Journal of Immunotoxicology 04/2012; 9(4). DOI:10.3109/1547691X.2012.664578 · 1.91 Impact Factor