Manchester D K, Gordon S K, Golas C L, Roberts E A, Okey A B
Cancer Res. 1987 Sep 15;47(18):4861-8.
Aryl hydrocarbon hydroxylase (AHH, cytochrome P1-450) is highly inducible in several human cells and tissues exposed to specific halogenated and nonhalogenated aromatic chemicals of the "3-methylcholanthrene-type." In laboratory animals AHH induction is known to be regulated by binding of inducers to the Ah receptor, a soluble intracellular protein. However, the induction mechanism in the human species is incompletely understood largely because the Ah receptor, which seems to be essential to the induction process, has not previously been detectable in certain human cells and tissues (including placenta) that are highly responsive to AHH induction. We found that human placenta contains high concentrations of Ah receptor (comparable to the receptor concentrations in rat and mouse liver) but that special modifications were necessary in the assay techniques in order to detect and accurately quantitate receptor binding. Receptor was detected at concentrations approximately equal to 100 fmol/mg cytosol protein using [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as the radioligand. This high concentration of specific binding sites was present only if the placental tissue was initially homogenized in a buffer containing sodium molybdate (10 or 20 mM). Without molybdate in the homogenizing buffer, specific [3H]TCDD binding was only about 35 fmol/mg. Specific Ah receptor binding also was detectable with [3H]-3-methylcholanthrene and, to a lesser extent, with [3H]-benzo(alpha)pyrene. The receptor sedimented near 9S on sucrose gradients whether molybdate was present or not. About 80% of specific binding was lost if excessive charcoal was used to adsorb "nonspecifically bound" ligand from cytosol prior to gradient analyses. The apparent affinity with which [3H]TCDD bound to Ah receptor in human placental cytosol was relatively low (apparent Kd approximately equal to 5 to 8 nM) when compared with the affinity of [3H]TCDD binding in rat or mouse hepatic cytosols (Kd approximately equal to 1 to 3 nM). These data suggest that while molybdate has very little effect on the quantity or molecular size of the rodent Ah receptor assay, it is very important in stabilizing the human Ah receptor. Our experiments demonstrate that human placenta contains a high concentration of Ah receptor and suggest that AHH induction in placenta is mediated through a receptor mechanism analogous to that previously established in tissues and cells from laboratory animals.
芳烃羟化酶（AHH，细胞色素P1 - 450）在暴露于“3 - 甲基胆蒽型”特定卤代和非卤代芳香族化学物质的几种人类细胞和组织中具有高度诱导性。在实验动物中，已知AHH诱导是由诱导剂与Ah受体（一种可溶性细胞内蛋白质）结合来调节的。然而，人类的诱导机制尚未完全了解，主要是因为在某些对AHH诱导高度敏感的人类细胞和组织（包括胎盘）中，此前一直无法检测到似乎对诱导过程至关重要的Ah受体。我们发现人类胎盘含有高浓度的Ah受体（与大鼠和小鼠肝脏中的受体浓度相当），但为了检测并准确定量受体结合，检测技术需要进行特殊改进。使用[³H]2,3,7,8 - 四氯二苯并 - p - 二恶英（TCDD）作为放射性配体，在浓度约为100 fmol/mg胞质溶胶蛋白时检测到了受体。只有当胎盘组织最初在含有钼酸钠（10或20 mM）的缓冲液中匀浆时，才会出现这种高浓度的特异性结合位点。如果匀浆缓冲液中没有钼酸钠，特异性[³H]TCDD结合量仅约为35 fmol/mg。用[³H] - 3 - 甲基胆蒽也可检测到特异性Ah受体结合，程度稍低的是用[³H] - 苯并(α)芘。无论是否存在钼酸钠，受体在蔗糖梯度上都沉降在9S附近。在进行梯度分析之前，如果使用过量活性炭从胞质溶胶中吸附“非特异性结合”的配体，约80%的特异性结合会丧失。与大鼠或小鼠肝脏胞质溶胶中[³H]TCDD结合的亲和力（Kd约为1至3 nM）相比，[³H]TCDD与人胎盘胞质溶胶中Ah受体结合的表观亲和力相对较低（表观Kd约为5至8 nM）。这些数据表明，虽然钼酸钠对啮齿动物Ah受体检测的数量或分子大小影响很小，但对稳定人类Ah受体非常重要。我们的实验表明人类胎盘含有高浓度的Ah受体，并表明胎盘中AHH诱导是通过一种类似于先前在实验动物组织和细胞中确立的受体机制介导的。
