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Ah Receptor in Human Placenta: Stabilization by Molybdate and Characterization of Binding of 2,3,7,8-Tetrachlorodibenzo-p-dioxin, 3-Methylcholanthrene, and Benzo(a)pyrene¹

Departments of Pediatrics and Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262 [D. K. M., S. K. G.] and Division of Clinical Pharmacology, Department of Pediatrics, Research Institute, The Hospital for Sick Children and Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada M5G 1X8 [C. L. G., E. A. R., A. B. O.]

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-methylcholan-threne-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 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(a)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 K_d 5 to 8 nM) when compared with the affinity of [³H]TCDD binding in rat or mouse hepatic cytosols (K_d 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.

¹ Supported by a Grant from the National Cancer Institute of Canada to A.B.O.

² Supported by NIH Short-term Training Grant TSAM07496.

³ To whom requests for reprints should be addressed, at the Division of Clinical Pharmacology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8.

Received 2/13/87. Revised 5/28/87. Accepted 6/24/87.

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