Roles of FMO and CYP450 in the metabolism in human liver microsomes of S-methyl-N,N-diethyldithiocarbamate, a disulfiram metabolite.
Roles of FMO and CYP450 in the metabolism in human liver microsomes of S-methyl-N,N-diethyldithiocarbamate, a disulfiram metabolite.
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DOI：
10.1111/j.1530-0277.1999.tb04274.x
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发表时间：
1999-07
期刊：
影响因子：
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通讯作者：
M. Pike;Y. Martin;D. Mays;L. Benson;Stephen Naylor;J. Lipsky
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文献类型：
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作者：
M. Pike;Y. Martin;D. Mays;L. Benson;Stephen Naylor;J. Lipsky
背景技术S-甲基-N,N-二乙基二硫代氨基甲酸酯(MeDDC)向MeDDC亚磺的转化是双硫仑(一种酒精威慑剂)代谢途径中甲基化后的第一步，其最终活性代谢物。最近已证明 CYP450 的各种亚型可以催化这种反应，但黄素单加氧酶 (FMO) 在人类这种代谢中的参与尚未得到评估。在本研究中，我们检测了昆虫微粒体中重组人FMO3代谢MeDDC的能力，并研究了FMO和CYP450在人肝微粒体中MeDDC代谢中的相对作用。方法采用高效液相色谱-质谱联用技术对人肝微粒体和重组人FMO3形成的MeDDC产物进行鉴定。通过使用热灭活来抑制 FMO，或使用 N-苯甲基咪唑 (NBI) 或 CYP450 NADPH 还原酶抗体来抑制 CYP450，研究了人肝微粒体中的 MeDDC 代谢。结果通过HPLC-质谱分析证实MeDDC硫磺酸是人肝微粒体与FMO3形成MeDDC的主要产物。重组 FMO3 是形成 MeDDC 硫磺的有效催化剂（5.3+/-0.2 nmol/min/mg，平均值+/-SEM，n = 6）。抑制研究表明，在 pH 7.4 的人肝微粒体中，MeDDC 主要通过 CYP450 代谢，其中 10% 来自 FMO（微粒体总活性 3.1+/-0.2，n = 17）。在这项工作过程中，发现甲基对甲苯硫醚 (MTS) 是人肝微粒体中 FMO 和 CYP450 的底物，一些研究人员将其磺化作用用作 FMO 活性的特异性探针。结论我们的结果证明MeDDC硫磺酸是人肝微粒体中MeDDC氧化的主要产物，MeDDC是人FMO3的良好底物，并且MeDDC在人肝微粒体中主要通过CYP450代谢。我们还表明，使用 MTS 磺化作用作为微粒体中 FMO 活性的指标仅在 CYP450 抑制剂（例如 NBI）存在的情况下才有效。
BACKGROUND The conversion of S-methyl-N,N-diethyldithiocarbamate (MeDDC) to MeDDC sulfine is the first step after methylation in the metabolic pathway of disulfiram, an alcohol deterrent, to its ultimate active metabolite. Various isoforms of CYP450 have recently been shown to catalyze this reaction, but the involvement of flavin monooxygenase (FMO) in this metabolism in humans has not been evaluated. In this study we examined the ability of recombinant human FMO3 in insect microsomes to metabolize MeDDC, and investigated the relative roles of FMO and CYP450 in the metabolism of MeDDC in human liver microsomes. METHODS HPLC-mass spectrometry was used to identify the products of MeDDC formed by human liver microsomes and by recombinant human FMO3. MeDDC metabolism in human liver microsomes was studied by using either heat inactivation to inhibit FMO, or N-benzylimidazole (NBI) or antibodies to the CYP450 NADPH reductase to inhibit CYP450. RESULTS We confirmed by HPLC-mass spectrometry that MeDDC sulfine was the major product of MeDDC formed by human liver microsomes and by FMO3. Recombinant FMO3 was an efficient catalyst for the formation of MeDDC sulfine (5.3+/-0.2 nmol/min/mg, mean+/-SEM, n = 6). Inhibition studies showed MeDDC was metabolized primarily by CYP450 in human liver microsomes at pH 7.4, with a 10% contribution from FMO (total microsomal activity 3.1+/-0.2, n = 17). In the course of this work, methyl p-tolyl sulfide (MTS), sulfoxidation of which is used by some investigators as a specific probe for FMO activity, was found to be a substrate for both FMO and CYP450 in human liver microsomes. CONCLUSIONS Our results prove that MeDDC sulfine is the major product of MeDDC oxidation in human liver microsomes, MeDDC is a good substrate for human FMO3, and MeDDC is metabolized in human liver microsomes primarily by CYP450. We also showed that use of MTS sulfoxidation as an indicator of FMO activity in microsomes is valid only in the presence of a CYP450 inhibitor, such as NBI.