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Division of Pharmaceutics, College of Pharmacy, The University of Iowa, Iowa City, Iowa (S.R., C.K.S.); Department of Plastic Surgery, Mercy Hospital, Iowa City, Iowa (A.E.C., A.A.); and Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy & Pharmaceutical Sciences, Purdue University, West Lafayette, Indiana (C.K.S.)
(Received March 2, 2007; Accepted June 5, 2007)
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Abstract |
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). Our current understanding of these reactions suggests that the cellular immune system plays a key role in precipitating CDRs. Bioactivation of the parent drug to its reactive metabolite, followed by protein haptenation, is probably a critical step in the elicitation of such reactions (Sanderson et al., 2006). Keratinocytes (KCs), the most abundant cell type in the epidermis, have been suggested to play a central role in the initiation and propagation of CDRs (Roychowdhury and Svensson, 2005). Indeed, cultured KCs and fibroblasts have been shown to bioactivate the sulfone dapsone (DDS) and sulfamethoxazole, leading to protein haptenation (Roychowdhury et al., 2005; Bhaiya et al., 2006). In addition, human dendritic cells can mediate this bioactivation (Roychowdhury et al., 2007). These drug-protein covalent adducts may act as neoantigens and trigger an immune response. Although the ability of various skin cells to metabolize sulfonamides or sulfones leading to protein haptenation has been established using isolated cell culture models, the relevance of these observations to events occurring in intact skin is unclear. To test the ability of skin to metabolize drugs leading to haptenation of cellular proteins, we exposed human organotypic skin explant cultures (hOSECs) to DDS or its hydroxylamine metabolite (D-NOH) and determined the formation of DDS-protein adducts using immunocytochemistry.
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Materials and Methods |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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) and determined by high-performance liquid chromatography to be >97% pure. Rabbit antisera were raised against DDS-keyhole limpet hemo-cyanin conjugates and specificity was assessed as described previously (Reilly et al., 2000). Goat-anti-rabbit IgG conjugated with Alexa Fluor 488 (Millipore Corporation, Billerica, MA) and goat anti-mouse IgG conjugated with Alexa Fluor 568 were purchased from Invitrogen (Carlsbad, CA). Anti-HLA-DR antibody was purchased from eBioscience (San Diego, CA). Immunomount was obtained from Vector Laboratories (Burlingame, CA). Penicillin, streptomycin, HEPES, and L-glutamine were purchased from Invitrogen. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Chicago, IL). Human Organotypic Skin Explant Cultures. Fresh human skin was obtained from patients undergoing abdominoplasty surgery and split-thickness skin samples (0.5 mm) were prepared using a dermatome. Skin samples were transported from the surgical suite to the laboratory in a sterile container on ice (within 1 h after excision). Skin was then cut into in 2- x 2-cm2 sections in a sterile hood and placed on Falcon cell culture inserts (BD Biosciences Discovery Labware, Bedford, MA; pore size, 1 µm) which, in turn, were placed inside six-well plates (one section per well). hOSECs were cultured at the air-medium interface with the epidermal side facing upward. Culture medium (1.5 ml per well) was poured carefully inside the wells, so that the bottom of the explants was just touching the medium and medium did not flow over the top of the explant. Culture medium was composed of 90% Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 100 units/ml penicillin and streptomycin, 1 mM L-glutamine, 1 mM HEPES, and 2-mercaptoethanol. hOSECs were then placed inside a humidified incubator at 37°C, 5% CO2 for 2 h followed by drug treatment.
Treatments. DDS (800 µM) or D-NOH (100 µM) was either mixed in the medium or applied (200 µl) on the epidermal surface of the skin and incubated for 24 h at 37°C with 5% CO2. At the end of the incubation period, hOSECs were washed three times with PBS, fixed in 10% buffered Formalin (2 days at room temperature), and dipped into 30% sucrose solution overnight at 4°C until explants sank to the bottom of the container. Sections were then embedded in optimal cutting temperature medium (OCT) and cryosectioned (5 µm thick).
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Results and Discussion |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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). Uptake of haptenated proteins by dendritic cells, followed by their migration to draining lymph nodes, is essential for the recruitment of a population of antigen-specific T cells to the skin. Limited evidence suggested that bioactivation of small molecules may occur within the skin itself, with the subsequent formation of neoantigens. We postulated that a similar mechanism may be responsible for the development of CDRs after systemic drug administration (Reilly et al., 2000). Although we have previously demonstrated the ability of cultured epidermal KCs (Roychowdhury et al., 2005), fibroblasts (Bhaiya et al., 2006), and dendritic cells (Roychowdhury et al., 2007) to bioactivate sulfamethoxazole and dapsone, giving rise to haptenated proteins, whether or not such metabolic activation occurs in intact skin is unclear.
Many contact-sensitizing agents, such as urushiol (the chemical in poison ivy), haptenate proteins after application to the skin (Kalish et al., 1994). Hence, our first effort to assess bioactivation and protein haptenation with skin exposed to DDS was performed by epicutaneous application of DDS or its metabolite. As shown in Fig. 1 (column 1), hOSECs exposed through this means gave rise to readily detected haptenated proteins. These haptenated proteins were mainly present in the uppermost epidermal layer of the skin with a low level of haptenation in the lower epidermal region. Although the therapeutic use of DDS is primarily via oral administration, topical application is also used (Draelos et al., 2007). Thus, our observations suggest that this route of application may give rise to DDS-protein conjugates in the skin.
To determine whether these haptenated proteins were formed in dendritic cells as well as KCs, we assessed whether haptenated proteins colocalized with HLA-DR (which is constitutively expressed on dendritic cells but not KCs). HLA-DR+ cells were readily detected by these hOSECs (Fig. 1, column 2), indicating that dendritic cells were retained in the explant for the duration of the incubation. Colocalization of dapsone-haptenated proteins (green) with HLA-DR (red) is evident by the orange region of double-stained explants (Fig. 1, column 3). Control cultures exposed to vehicle, DMSO, showed only HLA-DR staining with no haptenated proteins detected, as illustrated by the appearance of red color in the merged images.
When skin is exposed to xenobiotics after systemic administration, chemical will diffuse from the vascular bed through the hypodermal and into the epidermal region. To mimic this directional exposure, hOSECs were exposed to DDS or D-NOH by mixing drug/metabolite in the medium. Since medium did not flow over the top (epidermal region) of the explant, exposure to drug within the explant in this paradigm occurred from the "vascular" side. As shown in Fig. 2 (column 1), haptenated proteins were readily detected throughout the epidermal region of the explant when exposed to either DDS or D-NOH. In addition, drug/metabolite-haptenated proteins were evident in HLA-DR+ cells, indicating their presence in epidermal dendritic cells (Fig. 2, column 4). Experiments were conducted in two additional patient explant samples with similar results.
These results suggest that human skin is capable of bioactivating arylamine xenobiotics and may give rise to haptenated proteins. The presence of drug/metabolite-haptenated proteins in HLA-DR+ cells suggests either the transfer of KC-derived neoantigens or the haptenation of proteins in dendritic cells directly. Our previous work demonstrating the ability of cultured human dendritic cells to bioactivate DDS, giving rise to haptenated proteins, supports the latter explanation (Roychowdhury et al., 2007). In either case, an important next step will be to determine whether migrating dendritic cells from such explants exhibit haptenated proteins. Furthermore, these studies provide the basis for probing human skin biopsies from patients with CDRs for the presence of drug-haptenated proteins.
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Acknowledgments |
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Footnotes |
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ABBREVIATIONS: CDR, cutaneous drug reaction; DDS, dapsone; D-NOH, dapsone hydroxylamine; HLA, human leukocyte antigen; hOSEC, human organotypic skin explant culture; KC, keratinocyte; PBS, phosphate-buffered saline.
Address correspondence to: Dr. Craig K. Svensson, Office of the Dean, College of Pharmacy, Nursing and Health Sciences, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 49707. E-mail: svensson{at}purdue.edu
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  C. K. Svensson Biotransformation of Drugs in Human Skin Drug Metab. Dispos., February 1, 2009; 37(2): 247 - 253. [Abstract] [Full Text] [PDF]
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