Vyas Piyush M, Roychowdhury Sanjoy, Khan Farah D, Prisinzano Thomas E, Lamba Jatinder, Schuetz Erin G, Blaisdell Joyce, Goldstein Joyce A, Munson Kimber L, Hines Ronald N, Svensson Craig K
Division of Pharmaceutics, College of Pharmacy, University of Iowa, Iowa City, Iowa, USA.
J Pharmacol Exp Ther. 2006 Oct;319(1):488-96. doi: 10.1124/jpet.106.105858. Epub 2006 Jul 20.
Cutaneous drug reactions (CDRs) are among the most common adverse drug reactions and are responsible for numerous minor to life-threatening complications. Several arylamine drugs, such as sulfamethoxazole (SMX) and dapsone (DDS), undergo bioactivation, resulting in adduction to cellular proteins. These adducted proteins may initiate the immune response that ultimately results in a CDR. Recent studies have demonstrated that normal human epidermal keratinocytes (NHEKs) can bioactivate these drugs, resulting in protein haptenation. We sought to identify the enzyme(s) responsible for this bioactivation in NHEKs. Using immunofluorescence confocal microscopy and an adduct-specific enzyme-linked immunosorbent assay (ELISA), we found that N-acetylation of the primary amine of SMX and DDS markedly reduced the level of protein haptenation in NHEKs. Detection of mRNA and/or protein confirmed the presence of CYP3A4, CYP3A5, and CYP2E1 in NHEKs. In contrast, although a faint band suggestive of CYP2C9 protein was detected in one NHEK sample, a CYP2C9 message was not detectable. We also examined the ability of chemical inhibitors of cytochromes P450 (aminobenzotriazole and 1-dichloroethylene) and cyclooxygenase (indomethacin) to reduce protein haptenation when NHEKs were incubated with SMX or DDS by either confocal microscopy or ELISA. These inhibitors did not significantly attenuate protein adduction with either SMX or DDS, indicating that cytochromes P450 and cyclooxygenase do not play important roles in the bioactivation of these xenobiotics in NHEKs and thus suggesting the importance of other enzymes in these cells.
皮肤药物反应（CDR）是最常见的药物不良反应之一，可导致众多从轻微到危及生命的并发症。几种芳胺类药物，如磺胺甲恶唑（SMX）和氨苯砜（DDS），会发生生物活化，导致与细胞蛋白结合。这些结合的蛋白可能引发免疫反应，最终导致皮肤药物反应。最近的研究表明，正常人表皮角质形成细胞（NHEK）可以使这些药物发生生物活化，导致蛋白半抗原化。我们试图确定NHEK中负责这种生物活化的酶。使用免疫荧光共聚焦显微镜和加合物特异性酶联免疫吸附测定（ELISA），我们发现SMX和DDS伯胺的N-乙酰化显著降低了NHEK中的蛋白半抗原化水平。mRNA和/或蛋白检测证实NHEK中存在CYP3A4、CYP3A5和CYP2E1。相比之下，虽然在一个NHEK样本中检测到一条微弱的提示CYP2C9蛋白的条带，但未检测到CYP2C9信息。我们还通过共聚焦显微镜或ELISA检测了细胞色素P450（氨基苯并三唑和1-二氯乙烯）和环氧化酶（吲哚美辛）的化学抑制剂在NHEK与SMX或DDS孵育时减少蛋白半抗原化的能力。这些抑制剂并没有显著减弱SMX或DDS的蛋白加合，表明细胞色素P450和环氧化酶在NHEK中这些外源性物质的生物活化中不发挥重要作用，因此提示其他酶在这些细胞中的重要性。
