Alkoxyresorufin O-dealkylase assay using a rat hepatocyte spheroid microarray
Alkoxyresorufin O-dealkylase assay using a rat hepatocyte spheroid microarray
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DOI：
10.1016/j.jbiosc.2009.10.001
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发表时间：
2010-04-01
影响因子：
2.8
通讯作者：
Nakazawa, Kohji
中科院分区：
文献类型：
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作者：
Sakai, Yusuke;Tanaka, Tomoko;Nakazawa, Kohji
维持肝脏功能高表达的肝细胞多细胞聚集体(球体)被认为是一种有用的培养技术，可用于各种基于细胞的检测。在这项研究中，我们研究了肝细胞球体微阵列(HSM)芯片的药物代谢功能，该芯片包含672个原代大鼠肝细胞球体阵列，位于聚甲基丙烯酸甲酯平板(24×24 mm)中心100 mm(2)区域内，并使用烷氧基苯氧基异硫氰酸酯(乙氧基、甲氧基、戊氧基和苄氧基)O-脱烷基酶测定系统。在细胞色素P450酶的诱导剂3-甲基胆蒽(3-MC)的作用下，HSM芯片的乙氧基间苯二酚O-脱烷基酶(EROD)活性比单层肝细胞高5~10倍，至少可维持2周。我们还发现，3-MC可诱导HSM芯片中EROD、甲氧基间苯二酚O-脱烷基酶(MROD)和苯氧基间苯二酚O-脱烷基酶(Brod)活性，而苯巴比妥钠(P450诱导剂)可诱导PEOD、Brod、EROD和MROD活性。相关P450酶基因表达的增加证实了这些活性的诱导。这些结果表明，HSM芯片对P450诱导剂有很好的响应，并且功能维持了很长时间。因此，HSM芯片可能是利用肝细胞进行药物代谢分析的一个很有前途的细胞平台。(C)2009年，日本生物技术学会。版权所有。
Hepatocyte multicellular aggregates (spheroids), which maintain high expression of liver functions, have been advocated as a useful culture technique for various cell-based assays. In this study, we investigated the drug metabolic function of a hepatocyte spheroid microarray (HSM) chip, which contained an array of 672 spheroids of primary rat hepatocytes within a 100-mm(2) region in the center of a poly(methylmethacrylate) plate (24 x 24 mm) and used an alkoxyresorufin (ethoxy-, methoxy-, pentoxy- and benzyloxyresorufin) O-dealkylase assay system. Ethoxyresorufin O-dealkylase (EROD) activity of the HSM chip initiated by 3-methylcholanthrene (3-MC), an inducer of cytochrome P450 enzymes, was 5- to 10-fold higher than that of monolayer hepatocytes, with activity being maintained for at least 2 weeks. We also demonstrated that 3-MC induced EROD, methoxyresorufin O-dealkylase (MROD) and benzyloxyresorufin O-dealkylase (BROD) activities in the HSM chip, while sodium phenobarbital (P450 inducer) induced pentoxyresorufin O-dealkylase (PROD), BROD, EROD and MROD activities. Induction of these activities was confirmed by increased gene expression of the related P450 enzymes. These results showed that the HSM chip had a good response to P450 inducers and that function was maintained for long periods of time. The HSM chip therefore may be a promising cellular platform for drug metabolic assays using hepatocytes. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.