Peng Fu-Chuo, Chang Chiao-Chia, Yang Ching-Yao, Edwards Robert J, Doehmer Johannes
Institute of Toxicology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei, Taiwan.
Toxicology. 2006 Feb 1;218(2-3):172-85. doi: 10.1016/j.tox.2005.10.010. Epub 2005 Dec 5.
Human liver microsomes, supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms, and control and CYP3A4 cDNA-transfected V79 Chinese hamster cells were tested for their ability to metabolize territrem B (TRB) and territrem C (TRC). Two TRB metabolites, designated MB(2) and MB(4), and one TRC metabolite, designated MC, were formed by all of these preparations. Of the nine supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5), only those expressing CYP3A4 or CYP3A5 metabolized TRB and TRC. MB(2), MB(4), and MC were formed by CYP3A4 cDNA-transfected V79MZ Chinese hamster cells, but not by non-transfected cells. In order to investigate which CYP450 isoforms were responsible for MB(2), MB(4) and MC formation in human liver microsomal preparations, six isoform-specific chemical inhibitors (furafylline, sulfaphenazole, omeprazole, quinidine, ketoconazole, and diethyldithiocarbamate) and antibodies against CYP3A4 were used. MB(2), MB(4), and MC formation was markedly inhibited by ketoconazole, but less affected by quinidine and sulfaphenazole. Anti-CYP3A4 antibody markedly inhibited MB(2), MB(4), and MC formation and also 6 beta-hydroxytestosterone formation from testosterone. The CYP3A-dependent reaction of testosterone 6 beta-hydroxylation showed a high correlation with 4 beta-C hydroxylation of TRB (r(2)=0.97, P<0.0001), O-demethylation of TRB (r(2)=0.95, P<0.0001), and 4 beta-C hydroxylation of TRC (r(2)=0.99, P<0.0001). Immunoblotting and RT-PCR showed that CYP3A4 and CYP3A5 were expressed in all four human liver microsomal preparations tested (HLM1-HLM4). The amount of MB(2), MB(4), and MC formed using different HLM preparations was related to the 6 beta-testosterone hydroxylase activity of the preparations. However, the extent of MB(2), MB(4), and MC formation was not related to the age or gender of the person from whom the microsomal sample was prepared. It was therefore suggest that CYP3A4 and CYP3A5 are the major enzymes responsible for TRB and TRC metabolism by human liver microsomes.
对人肝微粒体、表达不同人CYP450同工型的杆状病毒转化昆虫细胞的超微粒体以及对照和CYP3A4 cDNA转染的V79中国仓鼠细胞进行了检测，以评估它们代谢震颤毒素B（TRB）和震颤毒素C（TRC）的能力。所有这些制剂都形成了两种TRB代谢物，分别命名为MB(2)和MB(4)，以及一种TRC代谢物，命名为MC。在表达不同人CYP450同工型（1A2、2A6、2B6、2C9、2C19、2D6、2E1、3A4和3A5）的杆状病毒转化昆虫细胞的九种超微粒体中，只有表达CYP3A4或CYP3A5的超微粒体能够代谢TRB和TRC。MB(2)、MB(4)和MC由CYP3A4 cDNA转染的V79MZ中国仓鼠细胞形成，但非转染细胞则不能形成。为了研究人肝微粒体制剂中哪些CYP450同工型负责MB(2)、MB(4)和MC的形成，使用了六种同工型特异性化学抑制剂（呋拉茶碱、磺胺苯吡唑、奥美拉唑、奎尼丁、酮康唑和二乙基二硫代氨基甲酸盐）以及抗CYP3A4抗体。酮康唑显著抑制了MB(2)、MB(4)和MC的形成，但奎尼丁和磺胺苯吡唑的影响较小。抗CYP3A4抗体显著抑制了MB(2)、MB(4)和MC的形成，也抑制了睾酮形成6β-羟基睾酮的过程。睾酮6β-羟基化的CYP3A依赖性反应与TRB的4β-C羟基化（r(2)=0.97，P<0.0001）、TRB的O-去甲基化（r(2)=0.95，P<0.0001）以及TRC的4β-C羟基化（r(2)=0.99，P<0.0001）高度相关。免疫印迹和RT-PCR显示，CYP3A4和CYP3A5在所检测的四种人肝微粒体制剂（HLM1-HLM4）中均有表达。使用不同HLM制剂形成的MB(2)、MB(4)和MC的量与制剂的6β-睾酮羟化酶活性相关。然而，MB(2)、MB(4)和MC的形成程度与制备微粒体样品的人的年龄或性别无关。因此，提示CYP3A4和CYP3A5是人肝微粒体代谢TRB和TRC的主要酶。
