Pignatello M A, Kauffman F C, Levin A A
Laboratory of Cellular and Biochemical Toxicology, Rutgers University, Piscataway, New Jersey 08854, USA.
Toxicol Appl Pharmacol. 1997 Feb;142(2):319-27. doi: 10.1006/taap.1996.8047.
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.
芳香维甲酸（E）-4-[2-(5,6,7,8-四氢-5,5,8,8-四甲基-2-萘基)-1-丙烯基]苯甲酸（TTNPB）作为致畸剂，在几个物种中以及在小鼠肢芽细胞培养中抑制软骨形成的效力比全反式维甲酸（tRA）高1000倍。研究了导致TTNPB效力的因素，包括与核维甲酸受体（RARs和RXRs）、胞质结合蛋白（CRABPs）的结合以及TTNPB的代谢情况。对于竞争性结合测定和饱和动力学，从用编码适当核受体或结合蛋白的cDNA转染的COS-1细胞中获得核溶胶或胞质溶胶部分。TTNPB与RARα、β和γ结合，解离常数在纳摩尔范围内；然而，这些结合亲和力比tRA低10倍。尽管TTNPB的亲和力很高，但仅与核受体的结合亲和力不太可能解释TTNPB的效力。TTNPB对CRABPs的结合亲和力明显低于tRA。TTNPB不与[3H]9-顺式维甲酸竞争结合RXRα、β或γ。小鼠肢芽细胞培养是一种用于维甲酸致畸作用的特征明确的模型，用于比较TTNPB和tRA的代谢情况。在用任何一种维甲酸处理的肢芽细胞培养物的培养基中，在72小时内TTNPB的消失明显比tRA慢。在细胞摄取实验中，两种维甲酸达到大致相等浓度；然而，TTNPB从肢芽细胞中消失的速度明显比tRA慢。总体而言，这些结果表明，与RARs的高亲和力结合、对CRABPs的低亲和力以及对代谢的抗性有助于TTNPB的效力。
