RXR Agonists Research Articles

Adipocyte fatty acid-binding protein (aP2), a newly identified LXR target gene, is induced by LXR agonists in human THP-1 cells

The liver X receptors (LXRalpha and LXRbeta), ligand-activated transcription factors, belong to the superfamily of nuclear hormone receptors and have been shown to play a major role in atherosclerosis by modulating cholesterol and triglyceride metabolism. In this report, we describe a novel LXR target, the adipocyte fatty acid binding protein (aP2), which plays an important role in fatty acid metabolism, adipocyte differentiation and atherosclerosis. While LXR agonists induce aP2 mRNA expression in human monocytes (THP-1 cells) and macrophages in a time- and concentration-dependent manner, they have no effect on aP2 expression in human adipocytes. The increase in aP2 mRNA level was additive when THP-1 cells were treated with LXR and PPARgamma agonists. Also, an RXR agonist induced aP2 expression in these cells. While no additive effect was observed with LXR and RXR agonists, additive effects were observed with RXR and PPARgamma agonists. GW9662, a potent PPARgamma antagonist, inhibited PPARgamma-induced aP2 expression without affecting LXR-mediated aP2 expression indicating the induction is mediated directly through LXR activation. Analysis of human aP2 promoter revealed a potential LXR response element (LXRE). Gel shift data showed that the LXRalpha/RXRalpha heterodimer bound to the LXRE motif in aP2 promoter in vitro in a sequence-specific manner. Deletion and mutation analyses of the proximal aP2 promoter confirm that this is a functional LXRE. These data indicate for the first time that human macrophage aP2 promoter is a direct target for the regulation by LXR/RXR heterodimers.

RXR–LXR heterodimer modulators for the potential treatment of dyslipidemia

A number of RXR agonists were synthesized and screened in functional assays. The synthesis and the structure–activity relationship (SAR) within the series of compounds will be presented. Some in vivo data in rodent models for dyslipidemia and diabetes will also be presented.

PPARS and Obesity

Welcome to this special issue of PPAR Research dedicated to “PPARs and Obesity.” Obesity and the interrelated disorders of the metabolic syndrome are a global health epidemic. To address this major problem, it is essential to understand the mechanisms regulating energy metabolism, and it has been known for years that PPARs play an important role in many facets of energy homeostasis. This is a very active and exciting field of research that, without a doubt, justifies a special issue. The genetic, molecular, and physiological aspects of PPARs as well as the metabolic effects of recently developed PPAR and RXR agonists are among the topics discussed. The issue begins with a review of key observations made in human subjects harboring genetic variations in PPARγ and a thorough overview of the metabolic effects of PPARs in genetically modified animal models. Over the last five years, the knowledge on PPAR delta biology has literally exploded and the potential therapeutic usefulness of this receptor in metabolic syndrome is now recognized. The interaction of PPARs with uncoupling proteins regulating energy expenditure is reviewed as are recent developments with RXR agonists. A closely related topic addresses the molecular and physiological functions of PPAR coactivators and corepressors in relationship to adipocyte energy metabolism. The selective PPAR modulator concept has attracted the attention of the field for over a decade; however the molecular bases underlying their differential mode of action have only begun to emerge recently. In addition to selective PPAR agonists, compounds that simultaneously activate two (dual agonists) or three (pan agonists) PPAR isoforms are in development. The potential advantages of these new combinations are discussed. The intriguing possibility that PPARs may mediate effects of caloric restriction on longevity is also considered. Finally, a growing body of evidence indicates that inflammation is a key feature of the obese state and that PPARs display strong anti-inflammatory properties. The evidence that PPARs may be interesting therapeutic targets to modulate obesity-induced inflammation is also reviewed. While these reviews just scratch the surface of PPAR/RXR interactions and regulation of energy balance, this special issue is packed with exciting, high quality reports from recognized experts in the field. We hope that the ideas presented here will generate further interest from the scientific community in this rapidly expanding area of research. Francine M. Gregoire Sander Kersten Wallace Harrington

Identification of Biomarkers Modulated by the Rexinoid LGD1069 (Bexarotene) in Human Breast Cells Using Oligonucleotide Arrays

Retinoids have been found to be promising chemopreventive agents that play an important role in regulating cell growth, differentiation, and apoptosis. The action of retinoids is mediated by retinoid receptors (retinoic acid receptors and retinoid X receptors), which are nuclear transcription factors that, when bound to retinoids, regulate gene expression. LGD1069 is a highly selective RXR agonist that has reduced toxicity compared with retinoids. Our previous studies have shown that RXR-selective ligands (or "rexinoids"), including LGD1069, can inhibit the growth of normal and malignant breast cells and can suppress the development of breast cancer in transgenic mice. For the current study, we attempted to identify biomarkers of the chemopreventive effect of the RXR-selective retinoid LGD1069. In these experiments, we used Affymetrix microarrays to identify target genes that were modulated by LGD1069 in normal human breast cells. Affymetrix and dChip analysis identified more than 100 genes that were up-regulated or down-regulated by LGD1069 treatment. We then tested 16 of these genes in validation experiments using quantitative reverse transcription-PCR and Western blotting of independently prepared samples, and found that 15 of 16 genes were modulated in a similar manner in these validation experiments as in the microarray experiments. Genes found to be regulated include known retinoid-regulated genes, growth regulatory genes, transcription factors, and differentiation markers. We then showed that the expression of several of these rexinoid-regulated biomarkers is modulated in vivo in mammary glands from mice treated with LGD1069. These critical growth-regulating proteins will be promising targets of future agents for the prevention and treatment of breast cancer.

Aza‐Retinoids as Novel Retinoid X Receptor‐Specific Agonists

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

The stimulatory effect of LXRα is blocked by SHP despite the presence of a LXRα binding site in the rabbit CYP7A1 promoter

The transcription of the cholesterol 7alpha-hydroxylase gene (CYP7A1) is greatly decreased in cholesterol-fed rabbits. To determine whether the molecular structure of the promoter is responsible for this downregulation, we cloned the rabbit CYP7A1 promoter, identified the binding sites for alpha-fetoprotein transcription factor (FTF) and liver X receptor (LXRalpha), and studied the effects of FTF, LXRalpha, and SHP on its transcription. Adding LXRalpha/retinoid X receptor together with their ligands (L/R) to the promoter/reporter construct transfected into HepG2 cells greatly increased its activity. FTF did not increase promoter activity, nor did it enhance the stimulatory effect of L/R. Mutating the FTF binding site abolished the promoter baseline activity. Increasing amounts of SHP abolished the effect of L/R, and FTF enhanced the ability of SHP to decrease promoter activity below baseline levels. Thus, downregulation of CYP7A1 in cholesterol-fed rabbits is attributable secondarily to the activation of farnesoid X receptor, which increases SHP expression to override the positive effects of LXRalpha. Although FTF is a competent factor for maintaining baseline activity, it does not further enhance and may suppress CYP7A1 transcription.

Retinol decreases beta‐catenin protein levels in retinoic acid (RA)‐resistant colon cancer cell lines via a RXR‐mediated degradation pathway

Excess nuclear beta-catenin induces colon cancer cell division. The objective of this study was to examine the effect of retinol on beta-catenin protein degradation. Three RA-resistant colon cancer cell lines were treated with 0, 0.1, 1 and 10 microM retinol for 1–4 d. Retinol reduced beta-catenin protein levels in a dose-responsive manner in all cell lines. Treatment with the proteasomal inhibitor, MG132, blocked the retinol-induced decrease in beta-catenin indicating retinol decreases beta-catenin via proteasomal degradation. Multiple pathways direct beta-catenin to the proteasome for degradation including a p53/Siah-1/adenomatous polyposis coli [APC], a Wnt/glycogen synthase kinase-3beta/APC, and a retinoid “X” receptor [RXR]-mediated pathway. Due to mutations in beta-catenin (HCT-116), APC (SW620), and p53 (WiDr) only the RXR-mediated pathway remains functional in all three cell lines. To test if RXRs mediate beta-catenin degradation, cells were treated with the RXR antagonist, PA452. PA452 blocked the retinol-induced decrease in beta-catenin protein. In contrast to retinol treatment, the RXR agonists, 9 cis-retinoic acid and PA024 only slightly reduced beta-catenin protein levels revealing that the RXR-mediated degradation pathway may not require a ligand-bound RXR. Decreased beta-catenin levels reflect growth inhibition in all three RA-resistant colon cancer cell lines indicating that retinol may regulate cell growth via a mechanism involving intracellular beta-catenin signaling. Funding: ACS Grant # 03-233-01-CNE.

Aza-retinoids as novel retinoid X receptor-specific agonists

A new structurally simple series of potent lipophilic aza-retinoids RXR agonists has been developed. SAR studies for the N-alkyl-azadienoic acids described here demonstrate that the RXR activity profile is sensitive to the N-alkyl chain length. Further, we have expanded the work to include azadienoic acids, which exhibited many accessible conformations leading to a better understanding of the SAR around the series.

Coactivation of Liver Receptor Homologue-1 by Peroxisome Proliferator-Activated Receptor γ Coactivator-1α on Aromatase Promoter II and Its Inhibition by Activated Retinoid X Receptor Suggest a Novel Target for Breast-Specific Antiestrogen Therapy

Aromatase inhibitors target the production of estrogen in breast adipose tissue, but in doing so, also decrease estrogen formation in bone and other sites, giving rise to deleterious side effects, such as bone loss and arthralgia. Thus, it would be clinically useful to selectively inhibit aromatase production in breast. In this regard, we have determined that the orphan nuclear receptor liver receptor homologue-1 (LRH-1) is a specific transcriptional activator of aromatase gene expression in human breast preadipocytes but not in other tissues of postmenopausal women. In this study, we show that the coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) is a physiologically relevant modulator of LRH-1, and that its transcriptional activity can be inhibited effectively using receptor-interacting peptide antagonists that prevent PGC-1alpha recruitment. Interestingly, we note that all of these peptides also interact in an agonist-dependent manner with retinoid X receptor alpha (RXRalpha), suggesting that these two receptors may compete for limiting cofactors within target cells. In support of this hypothesis, we show that 9-cis-retinoic acid, acting through RXR, inhibits both the basal and PGC-1alpha-induced transcriptional activity of LRH-1. The importance of this finding was confirmed by showing that LRH-1-dependent, PGC-1alpha-stimulated regulation of aromatase gene expression in primary human breast preadipocytes was effectively suppressed by RXR agonists. We infer from these data that LRH-1 is a bona fide target whose inhibition would selectively block aromatase expression in breast, while sparing other sites of expression.

NCoA4 Containing Transcriptional Complexes Modulates Response of Myelomonocytic Leukemic Cell Line to Retinoids and VD3.

Large scale analyses of transcriptome improve comprehension of complex processes such as differentiation or cell proliferation. SAGE libraries construction of the AML model U937 allowed the identification of new markers of myelomonocytic differentiation induced by retinoids and vitamin D3 (VD3) (Piquemal et al., 2002). Those molecules act through ligand-dependent transcription factors of the nuclear receptors family: RAR, RXR and VDR. Among differentially expressed members of transcriptional complexes, the most relevant was the co-regulator NCoA4 (Nuclear receptor Coactivator 4). This protein that modulates interactions between transcription factors, RNA polymerase II and chromatin remodeling factors, was initially described as a co-activator of the androgen receptor (AR). Its activity has been extended to receptors of estrogens (ER), peroxisome-proliferating activators (PPAR), retinoid X (RXR) and recently to VD3 receptor (VDR).Using real-time semi-quantitative PCR, we found that NCoA4 is specifically expressed during the monocytic differentiation of U937 but not during the granulocytic differentiation of NB4 cell lines. These results were confirmed by analysis on normal and in vitro-differentiated leukemia primary cells. Moreover, its early induction, within 6 hours of retinoids and VD3 treatment on U937 cells, suggests that its expression may be controlled by one or several nuclear receptors. Because of cross-talks between retinoids and VD3 pathways, we used NB4-LR2 cells in which RAR is knock-down. In this cell line, NCoA4 is expressed in a VDR-dependent fashion reinforcing the hypothesis that the coregulator is specifically involved in the VD3-monocytic differentiation of leukemic cells.Next, to explore the role of NCoA4, U937 cells were stably transfected to constitutively over-express the protein. The doubling time of this cell line (U-NCoA4) reaches to 48 hours against 24 hours in U937 cells. Concerning ligand-induced growth arrest, these cells are particularly sensitive to the RXR and VDR agonists while no significative difference was observed after treatment by the RAR agonist or by any combination. In addition, over-expression of NCoA4 induces a slow down of differentiation, as shown by expression of CD11b and CD14 myelomonocytic markers. Thus, in U-NCoA4, except for the RAR agonist, treatment for 72 hours corresponds to treatment for 48 hours levels of U937 cells.To conclude, in term of growth arrest, NCoA4 over-expressing cells are particularly sensitive to RXR and VDR agonists. Thus, natural ligands present in the culture medium might reduce or delay proliferation, inducing the same effect on differentiation. In order to have a large view of networks, transcriptome of U-NCoA4 was analyzed by real time PCR on Low Density Array composed of a hundred messengers extracted from U937 SAGE libraries. Analysis is currently in progress.

Differentiation and growth inhibition mediated via the RXR:PPARγ heterodimer in colon cancer

This study evaluated the anti-tumor efficacy of combining the RXR agonist, bexarotene, with the PPARγ agonist, rosiglitazone, in colon cancer. Moser, a human colon cancer cell line, was treated with bexarotene and rosiglitazone alone or in combination and the effect on growth and differentiation were examined. The data demonstrated that the bexarotene/rosiglitazone combination produced greater efficacy in growth inhibition than either single agent. Furthermore, combination treatment acted cooperatively to decrease COX-2 expression and PGE2 synthesis while increasing expression of the differentiation marker, CEA. These findings were confirmed in vivo in a Moser xenograft tumor model. Collectively, our data suggest a potential role for utilizing a combination regimen of a RXR and PPARγ agonist in the treatment of colon cancer.

Retinoid‐mediated stimulation of steroid sulfatase activity in myeloid leukemic cell lines requires RARα and RXR and involves the phosphoinositide 3‐kinase and ERK‐MAP kinase pathways

All-trans retinoic acid and 9-cis-retinoic acid stimulate the activity of steroid sulfatase in HL60 acute myeloid leukemia cells in a concentration- and time-dependent manner. Neither of these 'natural retinoids' augmented steroid sulfatase activity in a HL60 sub-line that expresses a dominant-negative retinoic acid receptor alpha (RARalpha). Experiments with synthetic RAR and RXR agonists and antagonists suggest that RARalpha/RXR heterodimers play a role in the retinoid-stimulated increase in steroid sulfatase activity. The retinoid-driven increase in steroid sulfatase activity was attenuated by inhibition of phospholipase D (PLD), but not by inhibitors of phospholipase C. Experiments with inhibitors of protein kinase C (PKC) show that PKCalpha and PKCdelta play an important role in modulating the retinoid-stimulation of steroid sulfatase activity in HL60 cells. Furthermore, we show that pharmacological inhibition of the RAF-1 and ERK MAP kinases blocked the retinoid-stimulated increase in steroid sulfatase activity in HL60 cells and, by contrast, inhibition of the p38-MAP kinase or JNK-MAP kinase had no effect. Pharmacological inhibitors of the phosphatidylinositol 3-kinase, Akt, and PDK-1 also abrogated the retinoid-stimulated increase in steroid sulfatase activity in HL60 cells. These results show that crosstalk between the retinoid-stimulated genomic and non-genomic pathways is necessary to increase steroid sulfatase activity in HL60 cells.

Trialkyltin Compounds Bind Retinoid X Receptor to Alter Human Placental Endocrine Functions

Retinoid X receptor (RXR) is a nuclear receptor that plays important and multiple roles in mammalian development and homeostasis. We previously reported that, in human choriocarcinoma cells, tributyltin chloride and triphenyltin hydroxide, which are typical environmental contaminants and cause masculinization in female mollusks, are potent stimulators of human chorionic gonadotropin production and aromatase activity, which play key endocrine functions in maintaining pregnancy and fetal development. However, the molecular mechanism through which these compounds stimulate these endocrine functions remains unclear. Our current study shows that trialkyltin compounds, including tributyltin chloride and triphenyltin hydroxide, function as RXR agonists. Trialkyltins directly bind to the ligand-binding domain of RXR with high affinity and function as transcriptional activators. Unlike the natural RXR ligand, 9-cis-retinoic acid, the activity of trialkyltins is RXR specific and does not activate the retinoic acid receptor pathway. In addition, trialkyltins activate RXR to stimulate the expression of a luciferase reporter gene containing the human placental promoter I.1 sequence of aromatase, suggesting that trialkyltins stimulate human placental endocrine functions through RXR-dependent signaling pathways. Therefore, our results suggest that activation of RXR may be a novel mechanism by which trialkyltins alter human endocrine functions.

Unraveling Protein-Protein Interactions in Living Cells with Fluorescence Fluctuation Brightness Analysis

Fluorescence correlation spectroscopy is a potentially powerful tool for measuring protein-protein interactions directly in single living cells. We previously reported on the detection of homodimer formation in cells using molecular brightness analysis. Here, we extend the technique to detect binding between different proteins. Proteins are labeled with the fluorescent markers YFP and CFP. We first determine the coexpression ratio of both proteins by measuring the intensity ratio with a dual-color setup. The effect of fluorescence resonance energy transfer on the intensity ratio is explicitly taken into account. The brightness of cells coexpressing both proteins is measured in a single-color setup. Selecting the laser wavelength of the two-photon light source allows us to either coexcite both proteins or to selectively excite YFP-labeled proteins. This approach enables us to distinguish between homodimer and heterodimer formation. We first present the theory and then demonstrate experimental feasibility using the ligand binding domains of retinoic acid receptor (RARLBD) and of retinoid X receptor (RXRLBD). Both proteins form heterodimers, and RXRLBD also forms homodimers in the presence of its agonist. We explore binding between these proteins in the presence and absence of RXR agonist. Our results demonstrate that brightness analysis offers a quantitative method for determining protein interactions in cells.

Synthesis, Crystal Structure Analysis, and Pharmacological Characterization of Disila-bexarotene, a Disila-Analogue of the RXR-Selective Retinoid Agonist Bexarotene

Twofold sila-substitution (C/Si exchange) in the saturated ring of the tetrahydronaphthalene skeleton of the RXR-selective retinoid agonist bexarotene (1a) leads to disila-bexarotene (1b). Compound 1b was synthesized in a multistep synthesis, starting from 1,2-bis(chlorodimethylsilyl)ethane. The identity of 1b was established by elemental analyses and multinuclear NMR studies, and the C/Si analogues 1a and 1b (and an intermediate in the synthesis of 1b) were structurally characterized by single-crystal X-ray diffraction. Furthermore, 1a and 1b were studied for their interaction with retinoid X receptors. Although the twofold sila-substitution of 1a resulted in significant differences in the molecular structures of 1a and 1b, disila-bexarotene (1b) was shown to be a highly potent RXR agonist.

Regulation of CYP26A1 expression by selective RAR and RXR agonists in human NB4 promyelocytic leukemia cells

All- trans retinoic acid (ATRA) can induce complete remission in acute promyelocytic leukemia (APL), but resistance to this treatment develops rapidly partly due to increased ATRA metabolism. Among the cytochrome P450s (CYPs) involved in ATRA metabolism, the ATRA-inducible cytochrome P450 26A1 (CYP26A1) is particularly active although the molecular mechanisms involved in its regulation are not well defined in the target leukemia cells. To study CYP26A1 expression and regulation in APL cells, we used the NB4 promyelocytic leukemia cell line. CYP26A1 constitutive expression was barely detectable in NB4 cells, but ATRA could induce high levels of CYP26A1 expression, which reached a maximum at 72 h. To further define CYP26A1 induction mechanisms in the NB4 leukemia cells, we used RARs and RXR selective agonists. The RARα agonist BMS753 could elicit maturation, as expected, but not CYP26A1 expression. Treatment with the RARβ agonist BMS641, or the RARβ/γ agonist BMS961, could not elicit maturation, as expected, nor induce CYP26A1 expression. Because CYP26A1 expression could not be induced by RAR ligands alone, NB4 cells were then co-treated with the RXR agonist BMS649. The RXR agonist alone could not induce CYP26A1 expression, nor in combination with either the RARβ agonist or the RARβ/γ agonist. However, the combination of the RXR agonist and the RARα agonist could elicit a marked induction of CYP26A1 expression. In conclusion, we have shown that CYP26A1 induction is not essential for the granulocytic maturation of NB4 leukemia cells, and that CYP26A1 induction requires the activation of both RARα and RXR in these cells.

9‐cis‐Retinoic Acid Analogues with Bulky Hydrophobic Rings: New RXR‐Selective Agonists.

AbstractFor Abstract see ChemInform Abstract in Full Text.

1α,25‐dihydroxyvitamin D3 stimulates steroid sulphatase activity in HL60 and NB4 acute myeloid leukaemia cell lines by different receptor‐mediated mechanisms

Steroid sulphatase is a key enzyme in the biosynthesis of bioactive estrogens and androgens from highly abundant inactive circulating sulphated steroid precursors. Little is known about how the expression/activity of this enzyme is regulated. In this article, we show that of 1alpha,25(OH)2D3 stimulates an increase steroid sulphatase activity in the HL60 myeloid leukaemic cell line that is inhibited by a specific nuclear VDR (VDRnuc) antagonist and unaffected by plasma membrane-associated vitamin D receptor (VDRmem) agonists and antagonists. 1alpha,25(OH)2D3-mediated up-regulation of steroid sulphatase activity in HL60 cells was augmented by RXR agonists, blocked by RXR-specific antagonists, and RAR specific agonists and antagonists had no effect. In contrast, the 1alpha,25(OH)2D3-mediated up-regulation of steroid sulphatase activity in the NB4 myeloid leukaemic cell line was unaffected by the specific VDRnuc and RXR antagonists, but was blocked by a VDRmem-specific antagonist and was increased by VDRmem-specific agonists. The findings reveal that VDRnuc-RXR-heterodimers play a key role in the 1alpha,25(OH)2D3-mediated up-regulation of steroid sulphatase activity in HL60 cells. However, in NB4 cells, VDRnuc-derived signals do not play an obligatory role, and non-genomic VDRmem-derived signals are important.

9- cis-Retinoic acid analogues with bulky hydrophobic rings: new RXR-selective agonists

Stille cross-coupling of aryltriflates 10 and dienylstannane 11, oxidation and Horner–Wadsworth–Emmons reaction afforded stereoselectively retinoates 15. Saponification provided the carboxylic acids 8a and 8b, retinoids that incorporate a bulky hydrophobic ring while preserving the 9- cis-geometry of the parent system. In contrast to the pan-RAR/RXR agonistic profile of the lower homologue of 8a, compound 7 (LG100567), retinoids 8 showed selective binding and transactivation of RXR, devoid of significant RAR activation. In PLB985 leukemia cells that require RXR agonists for differentiation compounds 8 induced maturation in the presence of the RAR-selective pan-agonist TTNPB; this effect was blocked by an RXR-selective antagonist.

Potentiation of the teratogenic effects induced by coadministration of retinoic acid or phytanic acid/phytol with synthetic retinoid receptor ligands

Previous studies in our laboratory identified retinoid-induced defects that are mediated by RAR-RXR heterodimerization using interaction of synthetic ligands selective for the retinoid receptors RAR and RXR in mice (Elmazar et al. 1997, Toxicol Appl Pharmacol 146:21-28; Elmazar et al. 2001, Toxicol Appl Pharmacol 170:2-9; Nau and Elmazar 1999, Handbook of experimental pharmacology, vol 139, Retinoids, Springer-Verlag, pp 465-487). The present study was designed to investigate whether these RAR-RXR heterodimer-mediated defects can be also induced by interactions of natural and synthetic ligands for retinoid receptors. A non-teratogenic dose of the natural RXR agonist phytanic acid (100 mg/kg orally) or its precursor phytol (500 mg/kg orally) was coadministered with a synthetic RARalpha-agonist (Am580; 5 mg/kg orally) to NMRI mice on day 8.25 of gestation (GD8.25). Furthermore, a non-teratogenic dose of the synthetic RXR agonist LGD1069 (20 mg/kg orally) was also coadministered with the natural RAR agonist, all- trans-retinoic acid (atRA, 20 mg/kg orally) or its precursor retinol (ROH, 50 mg/kg orally) to NMRI mice on GD8.25. The teratogenic outcome was scored in day-18 fetuses. The incidence of Am580-induced resorptions, spina bifida aperta, micrognathia, anotia, kidney hypoplasia, dilated bladder, undescended testis, atresia ani, short and absent tail, fused ribs and fetal weight retardation were potentiated by coadministration of phytanic acid or its precursor phytol. Am580-induced exencephaly and cleft palate, which were not potentiated by coadministration with the synthetic RXR agonists, were also not potentiated by coadministration with either phytanic acid or its precursor phytol. LGD1069 potentiated atRA- and ROH-induced resorption, exencephaly, spina bifida, aperta, ear anotia and microtia, macroglossia, kidney hypoplasia, undescended testis, atresia ani, tail defects and fetal weight retardation, but not cleft palate. These results suggest that synergistic teratogenesis can be induced by coadministration of a natural RXR ligand (phytanic acid) with a synthetic RAR agonist (Am580). Thus, certain potentially useful therapeutic agents or nutritional factors such as phytanic acid should be tested for teratogenic risk by coadministration with other retinoid receptor agonists.