Metabolic and molecular factors that contribute to the toxicity of the aromatic retinoid, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,5,8-tetramethyl-2-naphthylenyl)-1-propenyl] benzoic acid (TTNPB)

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. Binding to retinoid nuclear receptors (RARs and RXRs) and cytosolic binding proteins (CRABPs), ligand off-rates, differences in transcriptional activity of the ligand receptor complex and half life were investigated as factors contributing to the remarkable toxicity of TTNPB. Competitive binding affinities, dissociation constants and binding kinetics were determined for TTNPB and compared to those of tRA using recombinant RARs and RXRs expressed in COS-1 cells. TTNPB binds to RARα, β and γ with K_ds (nM) 10-fold less than those of tRA; however, affinities of TTNPB for CRABPI and CRABPII were 27- and 3.5-fold lower than those for tRA, respectively. TTNPB did not compete with [3H]9- cis RA for binding to RXRs α, β and γ. Displacement of [3H]TTNPB from the RARs occurred at a significantly faster rate than that of [3H]tRA.; Transactivation of a luciferase reporter gene driven by the tRA response element transfected into JEG-3 cells was comparable for TTNPB and tRA over a 24 hour period with EC₅₀s in the nanomolar range; however, after 72 hours, activation by TTNPB was greater than that of tRA as indicated by EC₅₀s and threshold for activation.; Mouse limb bud cell cultures, a well-characterized model for retinoid teratogenesis, were used to compare the metabolic stability and teratogenic effects of TTNPB and tRA. TTNPB metabolism was significantly slower than that of tRA. Liarozole, a 4-hydroxylase inhibitor of tRA, inhibited tRA metabolism about 10% and greatly increased inhibition of chondrogenesis (40-fold) in this system to a level that resembled the potency of TTNPB. It is concluded that the toxicity of TTNPB is related to multiple factors including; high affinity binding to RARs, lower affinity to CRABPs, more potent receptor activation and a longer half-life compared to that of tRA. The marked increase in toxicity of tRA, resulting from a slight inhibition of metabolism, to a level similar to that of TTNPB in mouse limb bud cells strongly suggests that a prolonged half-life may be the most significant factor contributing to the remarkable toxicity of TTNPB.