Cotreau M M, von Moltke L L, Beinfeld M C, Greenblatt D J
Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA.
J Pharmacol Toxicol Methods. 2000 Jan-Feb;43(1):41-54. doi: 10.1016/s1056-8719(00)00086-1.
Studies were conducted to characterize assays for the isolation and quantitation of rat cytochrome P450 (CYP) 3A isoforms from hepatic and intestinal tissues. Isolated intestinal microsomes were analyzed for their alkaline phosphatase activity and CYP 3A immunoreactivity. The involvement of CYP 3A in the in vitro hydroxylation of midazolam (MDZ) was also evaluated using isoform specific chemical and antibody inhibitors. The effect of glycerol (a common constituent of the microsomal reconstitution buffer) concentration on in vitro MDZ hydroxylation was also investigated. Additionally, to verify that the intestinal preparation was adequate for use in studies investigating the induction of CYP3A at the MRNA, protein, and catalytic activity within a single animal, a separate induction study was carried out with the CYP 3A inducer dexamethasone (DEX). A reverse transcription-polymerase chain reaction (RT-PCR) assay and a quantitative Western blotting method were used to reliably detect differences in CYP 3A mRNA and immunoreactivity between DEX- and vehicle (VH)-treated tissues. The in vitro hydroxylation of MDZ evaluated CYP 3A catalytic activity and identified increases in CYP 3A activity caused by DEX in comparison to VH. Collectively, these described techniques provide an experimental model to study xenobiotic induction of rat hepatic and intestinal CYP 3A from the molecular to the catalytic level in individual rats without the need for pooling of tissue.
开展了多项研究，以表征从肝脏和肠道组织中分离和定量大鼠细胞色素P450（CYP）3A亚型的分析方法。对分离出的肠道微粒体进行碱性磷酸酶活性和CYP 3A免疫反应性分析。还使用亚型特异性化学抑制剂和抗体抑制剂评估了CYP 3A在咪达唑仑（MDZ）体外羟化反应中的作用。此外，还研究了甘油（微粒体重组缓冲液的常见成分）浓度对体外MDZ羟化反应的影响。另外，为了验证肠道制剂是否足以用于在单只动物体内研究CYP3A在mRNA、蛋白质和催化活性水平上的诱导情况，用CYP 3A诱导剂地塞米松（DEX）进行了一项单独的诱导研究。采用逆转录-聚合酶链反应（RT-PCR）分析和定量蛋白质免疫印迹法可靠地检测DEX处理组和赋形剂（VH）处理组组织之间CYP 3A mRNA和免疫反应性的差异。MDZ的体外羟化反应评估了CYP 3A催化活性，并确定与VH相比，DEX可导致CYP 3A活性增加。总体而言，这些所述技术提供了一个实验模型，可在不进行组织汇集的情况下，在个体大鼠中从分子水平到催化水平研究外源性物质对大鼠肝脏和肠道CYP 3A的诱导作用。
