Distribution and induction of CYP3A1 and CYP3A2 in rat liver and extrahepatic tissues
Distribution and induction of CYP3A1 and CYP3A2 in rat liver and extrahepatic tissues
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DOI：
10.1016/0006-2952(95)02107-8
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发表时间：
1995-12-22
影响因子：
5.8
通讯作者：
Edwards, RJ
中科院分区：
文献类型：
--
作者：
Debri, K;Boobis, AR;Edwards, RJ
以前，我们已经表明，针对细胞色素P450酶的高度特异性抗体可以通过靶向在C-末端的5-氨基酸序列来产生。虽然大鼠CYP 3A 1和CYP 3A 2共享89%的氨基酸序列相似性，但它们的C-末端残基中有3/5不同。为了产生对每种形式具有特异性的抗体，用分别对应于CYP 3A 1和CYP 3A 2的C-末端的肽IITGS和文加免疫家兔。在酶联免疫吸附试验中，两种抗体均与用双烯醇酮16 α-腈（PCN）处理的大鼠的肝微粒体组分强烈结合。通过与IITGS孵育强烈抑制抗IITGS抗体的结合，但文加的有效性低60倍。相反，文加对抗VINGA抗体结合的抑制比IITGS有效100倍。使用免疫印迹也发现了类似的抗体结合抑制。使用抗IITGS抗体的免疫吸附产生了一个单一的蛋白质从溶解的肝微粒体组分从PCN处理的大鼠，这是公认的抗IITGS抗体。这两种抗体结合到单一的蛋白质在肝脏中增加后，与PCN治疗，但只有抗IITGS抗体识别蛋白质在肺，小肠，和肾脏的未处理和PCN治疗的大鼠。此外，两种抗体与未诱导和诱导大鼠的肝和肝外微粒体组分的结合显示两种抗体识别的蛋白质表达存在差异，进一步证明了抗体特异性。因此，抗IITGS和抗VINGA抗体的结合是相互排斥的，并且分别与其靶抗原CYP 3A 1和CYP 3A 2的特异性结合一致。免疫细胞化学法检测细胞色素P450 3A 1和3A 2的分布。在未处理动物的肝脏中，发现CYP 3A 1和CYP 3A 2均在小叶中心区表达。然而，在整个小叶的许多（但不是全部）肝细胞中也检测到一些CYP 3A 1免疫反应性。然而，在用PCN处理大鼠后，发现CYP 3A 1和CYP 3A 2均在整个小叶的肝细胞中强烈表达，尽管CYP 3A 2在小叶中心区域显示出更高的表达。还发现PCN给药可诱导小肠、肺和肾脏特定区域的CYP 3A 1。
Previously, we have shown that highly specific antibodies against cytochrome P450 enzymes can be produced by targeting a 5-amino acid sequence at the C-terminus. Although rat CYP3A1 and CYP3A2 share 89% amino acid sequence similarity, they differ by 3 out of 5 of their C-terminal residues. In an effort to produce antibodies specific to each form, rabbits were immunised with the peptides IITGS and VINGA, corresponding to the C-termini of CYP3A1 and CYP3A2, respectively. Both antibodies bound strongly to hepatic microsomal fraction from rats treated with pregnenolone 16 alpha-carbonitrile (PCN) in enzyme-linked immunosorbent assay. Binding of the anti-IITGS antibody was strongly inhibited by incubation with IITGS, but VINGA was 60 times less effective. Conversely, binding of the anti-VINGA antibody was inhibited by VINGA 100 times more effectively than IITGS. Similar inhibition of antibody binding was also found using immunoblotting. Immunoadsorption using the anti-IITGS antibody yielded a single protein from solubilised hepatic microsomal fraction from PCN-treated rats, which was recognised only by the anti-IITGS antibody. Both antibodies bound to single proteins in the liver which were increased following treatment with PCN, but only the anti-IITGS antibody recognised protein in the lung, small intestine, and kidney of untreated and PCN-treated rats. Also, the binding of the two antibodies to hepatic and extrahepatic microsomal fractions from uninduced and induced rats showed differences in the expression of proteins recognised by the two antibodies, providing further evidence of antibody specificity. Thus, the binding of anti-IITGS and anti-VINGA antibodies is mutually exclusive and consistent with specific binding to their target antigens, CYP3A1 and CYP3A2, respectively. Immunocytochemistry was used to determine the distribution of CYP3A1 and CYP3A2. In the liver of untreated animals, both CYP3A1 and CYP3A2 were found to be expressed in the centrilobular region. However, some CYP3A1 immunoreactivity was also detected in many, but not all, hepatocytes throughout the lobule. However, following treatment of rats with PCN, both CYP3A1 and CYP3A2 were found to be strongly expressed in hepatocytes throughout the lobule, although CYP3A2 showed greater expression in the centrilobular region. PCN treatment was also found to result in induction of CYP3A1 in specific regions of the small intestine, lung, and kidney.