Multiple factors contribute to the toxicity of the aromatic retinoid TTNPB(Ro 13-7410): Interactions with the retinoic acid receptors

The aromatic retinoid, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-n aphthylenyl)-1-propenyl] benzoic acid (TTNPB) is 1000-fold more teratogenic than all trans-retinoic acid (tRA) in several species. Factors that partia lly explain the potency of this retinoid include binding affinities to reti noid nuclear receptors (RARs) in the nanomolar range, reduced affinities fo r the cytosolic binding proteins (CRABPs), and slow rate of metabolism (M. A. Pignatello, F. C. Kauffman, and A. A. Levin, Toxicol. Appl. Pharmacol. 1 42, 319-327, 1997). The present work investigates the possible involvement of longer receptor occupancy and increased transcriptional activity of the ligand receptor complex in the greater toxicity of TTNPB. Ligand off-rates from nuclear receptors were determined in nucleosol fractions prepared from COS-1 cells transfected with cDNA encoding the appropriate RAR subtype. Wh en assayed at 10 degrees C, [H-3]TTNPB was displaced from the RARs at a sig nificantly faster rate than that of [3H]tRA. The difference in displacement was reduced at 4 degrees C. These observations are consistent with the 10- fold lower affinity of TTNPB vs tRA for RARs and, therefore, do not explain the greater potency of TTNPB. The ability of TTNPB and tRA to activate the RARs was determined using a luciferase reporter gene transfected into JEG- 3 cells with the appropriate RAR subtype. The expression of the reporter wa s driven by a retinoic acid response element (RARE) from the RAR beta gene, which was incorporated into the reporter plasmid. Dose-response for gene a ctivation indicated that the potency of TTNPB and tRA in activating mRAR al pha, beta, and gamma was similar after 24 h with comparable EC50s in the na nomolar range. However, after 72 h, activation by TTNPB was greater than th at of tRA as indicated by EC50s and threshold for activation. This study in dicates that the higher potency of TTNPB in activating the RARs may be due to slower disappearance of the retinoid and, therefore, is a contributing f actor to its greater toxicity, (C) 1999 Academic Press.