Biochemical Pharmacology, Vol. 38, No. 15, pp. 2551-2557, 1989. Printed in Great Britain. NW.-2952/89 S3.00 + 0.00 Maxwell Pergamon Macmillan plc THE EFFECT OF THE PLASTICIZERS TBEP (TRIS-(2-BUTOXYETHYL)-PHOSPHATE) AND DEHP (DI-(2-ETHYLHEXYL)-PHTHALATE) ON P_ADRENERGIC LIGAND BINDING TO q-ACID GLYCOPROTEIN AND MONONUCLEAR LEUKOCYTES* GEORG SAGER and CLIVE LIITLE? Department of Pharmacology and tDepartment of Biochemistry, Institute of Medical Biology, P.O. Box 977, University of Tromse, N-9001 Troms0, Norway zyxwvutsrqponmlkjihgfedcbaZYX (Received 7 June 1988; accepted 14 February 1989) zyxwvutsrqponmlkjihgfedcbaZYXW Abstract-The plasticizers tris-(2-butoxyethyl)-phosphate (TBEP) and di-(2-ethylhexyl)-phthalate (DEHP) and the /I-adrenergic receptor-blockers [‘HI-(-)-dihydroalprenolol ([‘HI-(-)-DHA) and [‘HI- (-)-CGP 12177 were tested for their ability to interact with badrenergic binding to q-acid glycoprotein (AAG) and mononuclear leukocytes (MNL). The Ic5,-values, obtained by displacement of [3H]-(-)- DHA bound to AAG, were 3.5 nM, 2 PM and 4 PM for TBEP, (-)-alprenolol and DEHP, respectively. (-t)-CGP 12177 had virtually no effect on radioligand binding to AAG. The [3H]-(-)-CGP 12177 binding to MNL consisted of padrenergic receptor binding (& = 210 PM) and non-saturable binding. [ 3H]-( -)-DHA was bound to two different classes of binding sites on MNL, the Badrenergic receptors (& = 440 PM) and a secondary class of binding sites (& = 64 nM). (+)-CGP 12177 displaced about 30% of [3H]-( -)-DHA from MNL with an IcSO-valueof 190 pm. (-)-ALP displaced about 85% of total bound radioligand and gave a biphasic displacement curve with IcsO-values of 320pM and 69OmM, respectively. TBEP displaced a considerable fraction of t3H]-( -)-CGP 12177 and [3H]-( -)-DHA bound to MNL j?-adrenergic receptors, whereas DEHP had no effect. In contrast, DEHP caused displacement of [3H]-( -)-DHA from the MNL low affinity sites, but was a markedly less potent displacer compared to TBEP. The present study shows that TBEP and DEHP interact with padrenergic transport proteins, non-specific tissue binding sites and padrenergic receptors coupled to adenylate cyclase. Plasticizers may thus affect the biology and pharmacology of the padrenergic signal system. The binding sites of AAG and the padrenergic ducted primarily to determine whether TBEP and receptor have some properties in common. Both DEHP affect the padrenergic ligand binding to MNL padrenergic receptors and AAG possess a MNL in the same way as that to AAG. Secondly, binding site characterized by stereospecificity, and this study was undertaken to characterize the effect with higher binding affinity for antagonists than for of TBEP and DEHP on the different classes of MNL the agonists [l-6]. padrenergic binding sites. The cellular binding of Padrenergic ligands com- prises specific (receptor) binding and non-specific binding. The fraction of the hydrophilic radioligand [3H]-( -)-CGP 12177 non-specifically bound to intact MNL is small [7]. In contrast, the non-specific bind- ing of the lipophilic radioligand [3H]-( -)-DHA con- sists of saturable low affinity binding in addition to the non-saturable binding [l, 8,9]. However, the nature of the low affinity class of MNL [3H]-(-)- DHA binding sites is unknown, but it has been suggested that these binding sites may represent AAG adsorbed to the cells [9]. MATERIALS AND METHODS The plasticizers TBEP and DEHP cause a pro- nounced and selective displacement of basic drugs bound to AAG [lO-131. The present work was con- Chemicals. The following radiolabelled substances were used: (-)-4-(3-t-butylamino-2-hydroxypro- poxy)-[5,7-3H]-benzimidazol-2-one with sp. act. 44 Ci/mmol and (-)-[ propyl-2,3-3H]-dihydro- alprenolol with a sp. act. 38Ci/mmol, both from Amersham International (Bucks, U.K.). The purity of the radiolabelled ligands was verified by thin layer chromatography (TLC). The following unlabelled compounds were employed: (-)-alprenolol (+)-tar- trate from Sigma Chemical Co. (St Louis, MO), (k) - 4 - (3 - t - butylamino - 2 - hydroxypropoxy) - 1,3 - dihydrobenzimidazol-2-one hydrochloride from Ciba-Geigy AG (Basle, Switzerland), di-(Zethyl- hexyl)-phthalate from Aldrich-Chemie (Steinheim, F.R.G.), tris-(2-butoxyethyl)-phosphate from Aldrich Chemical Co. (Milwaukee, WI). All other reagents were of analytical grade. * Abbreviations: [‘HI-(-)-DHA, (-)-[propyl-2,3-‘HI- dihydroalprenolol; [3H]-(-)-CGP 12177, (-)-4-(3-t- butylamino-2-hydroxyropoxy)-[5,7-3H]-benzimidazol-2- one; (-c)-CGP 12177, (+)-4-(3-t-butylamino-2-hydroxy- propoxy)-1,3-dihydrobenzimidazol-2-one; (-)-ALP, (-)- alprenolol; AAG, q-acid glycoprotein; MNL, mono- nuclear leukocytes; TBEP, tris-(2-butoxyethyl)-phosphate; DEHP, di-(2_ethylhexyl)-phthalate. Zncubntion buffer. The medium comprised (mM): NaCl 122, KCL4.9, MgS04 1.2, CaC12 1.3 and Na2HP04 15.9, pH 7.38. AAG. AAG was purified from fresh human serum 2551