Rodriguez R J, Acosta D
Division of Pharmacology and Toxicology, College of Pharmacy, University of Cincinnati, OH 45267, USA.
Toxicology. 1997 Feb 28;117(2-3):123-31. doi: 10.1016/s0300-483x(96)03560-3.
Ketoconazole (KT) is an azole antifungal agent that has been associated with hepatotoxicity. The mechanism of its hepatotoxicity has not yet been resolved. It has been suggested that a reactive metabolite may be the cause of toxicity because the hepatic injury does not appear to be mediated through an immunoallergic mechanism. Several metabolites of KT have been reported in the literature of which the deacetylated metabolite, N-deacetyl ketoconazole (DAK), is the major metabolite which undergoes further metabolism by the flavin-containing monooxygenases (FMO) to form a potentially toxic dialdehyde. The objective of this study was to evaluate DAK's cytotoxicity and the role of FMO in a primary culture system of rat hepatocytes. Cytotoxicity was evaluated by measuring the leakage of the cytosolic enzyme, lactate dehydrogenase (LDH), into the medium and by assessing mitochondrial reduction of 3-(4,5-dimethythiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT). The cultures were exposed to various concentrations of DAK (20-160 microM) for 0.5-4 h. There was a significant increase (P < 0.05) in LDH leakage and an immediate decrease in MTT reduction (P < 0.05) as early as 0.5 h. The MTT reduction assay appeared to be more sensitive than the LDH assay in that lower concentrations were needed to observe a 50% reduction of MTT (107, 90, 75, 58 microM DAK at 0.5, 1.0, 2.0 and 4.0 h, respectively). The concentrations to observe 50% LDH leakage from the hepatocytes were 155, 133, 100, 70 microM DAK at 0.5, 1.0, 2.0 and 4.0 h, respectively. Moreover, co-treatment with methimazole, a competitive substrate for FMO, produced a significant decrease (P < 0.05) in % LDH leakage as early as 0.5 h, when compared to cells treated solely with DAK. Also, the toxicity was significantly (P < 0.05) enhanced as early as 0.5 h by n-octylamine, a known positive effector for FMO. These results demonstrate that DAK is a more potent cytotoxicant than its parent compound, KT, as reported previously by our laboratory (Rodriguez and Acosta, Toxicology, 96: 83-92, 1995) and its toxicity was expressed in a dose- and time-dependent manner. Furthermore, DAK's cytotoxicity was enhanced with n-octylamine and suppressed with methimazole, suggesting a role for FMO in the toxicity of the metabolite.
酮康唑（KT）是一种唑类抗真菌剂，与肝毒性有关。其肝毒性机制尚未明确。有人提出，一种反应性代谢产物可能是毒性的原因，因为肝损伤似乎不是通过免疫过敏机制介导的。文献中报道了KT的几种代谢产物，其中脱乙酰代谢产物N - 去乙酰酮康唑（DAK）是主要代谢产物，它通过含黄素单加氧酶（FMO）进一步代谢形成潜在有毒的二醛。本研究的目的是评估DAK在大鼠肝细胞原代培养系统中的细胞毒性以及FMO的作用。通过测量胞质酶乳酸脱氢酶（LDH）泄漏到培养基中的情况以及评估3 -（4,5 - 二甲基噻唑 - 2 - 基）- 2,5 - 二苯基溴化四氮唑（MTT）的线粒体还原情况来评估细胞毒性。将培养物暴露于不同浓度的DAK（20 - 160 microM）0.5 - 4小时。早在0.5小时，LDH泄漏就显著增加（P < 0.05），MTT还原立即减少（P < 0.05）。MTT还原测定似乎比LDH测定更敏感，因为观察到MTT减少50%所需的浓度更低（分别在0.5、1.0、2.0和4.0小时时为107、90、75、58 microM DAK）。观察到肝细胞50% LDH泄漏的浓度分别在0.5、1.0、2.0和4.0小时时为155、133、100、70 microM DAK。此外，与FMO的竞争性底物甲巯咪唑共同处理，与仅用DAK处理的细胞相比，早在0.5小时时LDH泄漏百分比就显著降低（P < 0.05）。而且，已知的FMO阳性效应物正辛胺早在0.5小时时就显著增强了毒性（P < 0.05）。这些结果表明，如我们实验室先前报道（Rodriguez和Acosta，《毒理学》，96：83 - 92，1995），DAK是一种比其母体化合物KT更有效的细胞毒性剂，其毒性呈剂量和时间依赖性表达。此外，DAK的细胞毒性被正辛胺增强，被甲巯咪唑抑制，表明FMO在该代谢产物的毒性中起作用。
