Roberts E A, Johnson K C, Harper P A, Okey A B
Division of Gastroenterology, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Arch Biochem Biophys. 1990 Feb 1;276(2):442-50. doi: 10.1016/0003-9861(90)90743-i.
The Ah receptor, a soluble cytoplasmic receptor that regulates induction of cytochrome P450IA1 and mediates toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was detected and characterized in the continuous human liver cell line Hep G2. The mean concentration of specific binding sites for TCDD was 112 +/- 26 (SEM) fmol/mg cytosol protein as determined in eight separate cytosol preparations in the presence of sodium molybdate. This is equivalent to 14,000 binding sites per cell, approximately 40% of the sites per cell found in the mouse hepatoma line Hepa-1. The cytosolic Ah receptor from Hep G2 cells sedimented at 9 S and was specific for those halogenated and nonhalogenated aromatic compounds known to be agonists for the Ah receptor in rodent tissues and cells. Specific binding in the 9 S region was detected with both [3H]TCDD and 3-[3H]methylcholanthrene. 3-[3H]Methylcholanthrene did not bind to any component besides that at approximately 9 S. Phenobarbital, dexamethasone, and estradiol did not compete with [3H]TCDD for binding to the Hep G2 Ah receptor. Specific binding of [3H]triamcinolone acetonide to glucocorticoid receptor could also be demonstrated in Hep G2 cytosol. The apparent equilibrium dissociation constant (Kd) for binding of [3H]TCDD to Hep G2 Ah receptor was 9 nM by Woolf plot analysis, about an order of magnitude weaker than the affinity of [3H]TCDD for the mouse Hepa-1 Ah receptor or for the C57BL/6 murine hepatic Ah receptor. [3H]TCDD.Ah receptor complex, which was extracted from nuclei of Hep G2 cells incubated with [3H]TCDD at 37 degrees C in culture, sedimented at approximately 6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was significantly induced after 24 h of incubation with polycyclic aromatic hydrocarbons: the EC50 for AHH induction was 5.3 microM for benz(a)anthracene and 1.3 microM for 3-methylcholanthrene. Modification of the preparative technique for cell cytosol, especially inclusion of 20 mM sodium molybdate in homogenizing and other buffers, was necessary to detect cytosolic Hep G2 Ah receptor. Hep G2 cells appear to conserve drug-metabolizing activity associated with cytochrome P450IA1 as well as the receptor mechanism which regulates its induction.
芳烃受体是一种可溶性胞质受体，可调节细胞色素P450IA1的诱导并介导2,3,7,8-四氯二苯并 - 对二恶英（TCDD）的毒性作用。在连续的人肝癌细胞系Hep G2中检测并鉴定了该受体。在存在钼酸钠的情况下，对八个单独的胞质溶胶制剂进行测定，TCDD特异性结合位点的平均浓度为112±26（SEM）fmol/mg胞质溶胶蛋白。这相当于每个细胞有14,000个结合位点，约为小鼠肝癌细胞系Hepa-1中每个细胞结合位点数的40%。Hep G2细胞的胞质芳烃受体在9S处沉降，对那些已知在啮齿动物组织和细胞中作为芳烃受体激动剂的卤代和非卤代芳香化合物具有特异性。用[3H]TCDD和3-[3H]甲基胆蒽均可检测到9S区域的特异性结合。3-[3H]甲基胆蒽除了在约9S处的成分外，不与任何其他成分结合。苯巴比妥、地塞米松和雌二醇不与[3H]TCDD竞争结合Hep G2芳烃受体。在Hep G2胞质溶胶中也可证明[3H]曲安奈德与糖皮质激素受体的特异性结合。通过伍尔夫图分析，[3H]TCDD与Hep G2芳烃受体结合的表观平衡解离常数（Kd）为9 nM，比[3H]TCDD对小鼠Hepa-1芳烃受体或C57BL/6小鼠肝脏芳烃受体的亲和力弱约一个数量级。在37℃培养条件下，用[3H]TCDD孵育Hep G2细胞的细胞核后提取的[3H]TCDD.芳烃受体复合物，在高离子强度条件下在约6S处沉降。与多环芳烃孵育24小时后，芳烃羟化酶（AHH）活性显著诱导：苯并(a)蒽诱导AHH的EC50为5.3μM，3-甲基胆蒽为1.3μM。为了检测胞质Hep G2芳烃受体，必须改进细胞胞质溶胶的制备技术，特别是在匀浆和其他缓冲液中加入20mM钼酸钠。Hep G2细胞似乎保留了与细胞色素P450IA1相关的药物代谢活性以及调节其诱导的受体机制。
