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-naphthylenyl)-1 -propenyl] benzoic acid (TTNPB) is 1000-fold more teratogenic than all trans-retinoic acid (tRA) in several species. Factors that partially explain the potency of this retinoid include binding affinities to retinoid nuclear receptors (RARs) in the nanomolar range, reduced affinities for the cytosolic binding proteins (CRABPs), and slow rate of metabolism (M. A. Pignatello, F. C. Kauffman, and A. A. Levin, Toxicol. Appl. Pharmacol. 142, 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. When assayed at 10 degrees C, [3H]TTNPB was displaced from the RARs at a significantly 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 was driven by a retinoic acid response element (RARE) from the RAR beta gene, which was incorporated into the reporter plasmid. Dose-response for gene activation indicated that the potency of TTNPB and tRA in activating mRAR alpha, beta, and gamma was similar after 24 h with comparable EC50s in the nanomolar range. However, after 72 h, activation by TTNPB was greater than that of tRA as indicated by EC50s and threshold for activation. This study indicates that the higher potency of TTNPB in activating the RARs may be due to slower disappearance of the retinoid and, therefore, is a contributing factor to its greater toxicity.

Multiple factors contribute to the toxicity of the aromatic retinoid, TTNPB (Ro 13-7410): binding affinities and disposition.

The aromatic retinoid (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1 -propenyl] benzoic acid (TTNPB) is 1000-fold more potent as a teratogen than all trans-retinoic acid (tRA) in several species and in the inhibition of chondrogenesis in the mouse limb bud cell culture. Factors responsible for the potency of TTNPB were investigated including binding to nuclear retinoic acid receptors (RARs and RXRs), cytosolic binding proteins (CRABPs), and metabolic disposition of TTNPB. For competitive binding assays and saturation kinetics, nucleosol or cytosol fractions were obtained from COS-1 cells transfected with cDNAs encoding the appropriate nuclear receptor or binding protein. TTNPB binds to RAR alpha, beta, and gamma with Kds in the nanomolar range; however, these binding affinities are 10-fold less than those of tRA. Although the affinities are high for TTNPB, it is unlikely that the binding affinities to nuclear receptors alone account for the potency of TTNPB. The binding affinities of TTNPB for the CRABPs are significantly lower than those of tRA. TTNPB did not compete with [3H]9-cis RA for binding to RXR alpha, beta, or gamma. Mouse limb bud cell cultures, a well characterized model for retinoid teratogenesis, were used to compare the metabolic disposition of TTNPB and tRA. In the media of limb bud cell cultures treated with either retinoid, the disappearance of TTNPB was significantly slower than that of tRA over 72 hr. Both retinoids reached approximately equal concentrations in cell uptake experiments; however, TTNPB disappeared from the limb bud cell at a significantly slower rate than did tRA. Collectively, these results indicate that high affinity binding to RARs, lower affinity to CRABPs, and resistance to metabolism contribute to the potency of TTNPB.