Understanding the mechanisms underlying alcohol metabolism and its regulation, including the effect of polymorphisms in alcohol-metabolizing enzymes, is crucial for research on Fetal Alcohol Spectrum Disorders. The aim of this study was to identify specific single nucleotide polymorphisms in key alcohol-metabolizing enzymes in a cohort of 71 children, including children with fetal alcohol syndrome, children prenatally exposed to ethanol but without fetal alcohol spectrum disorder, and controls. We hypothesized that certain genetic variants related to alcohol metabolism may be fixed in these populations, giving them a particular alcohol metabolism profile. In addition, the difference in certain isoforms of these enzymes determines their affinity for alcohol, which also affects the metabolism of retinoic acid, which is key to the proper development of the central nervous system. Our results showed that children prenatally exposed to ethanol without fetal alcohol spectrum disorder traits had a higher frequency of the ADH1B*3 and ADH1C*1 alleles, which are associated with increased alcohol metabolism and therefore a protective factor against circulating alcohol in the fetus after maternal drinking, compared to FAS children who had an allele with a lower affinity for alcohol. This study also revealed the presence of an ADH4 variant in the FAS population that binds weakly to the teratogen, allowing increased circulation of the toxic agent and direct induction of developmental abnormalities in the fetus. However, both groups showed dysregulation in the expression of genes related to the retinoic acid pathway, such as retinoic acid receptor and retinoid X receptor, which are involved in the development, regeneration, and maintenance of the nervous system. These findings highlight the importance of understanding the interplay between alcohol metabolism, the retinoic acid pathway and genetic factors in the development of fetal alcohol syndrome.

The methods described in this chapter concern procedures for the design, synthesis, and in vitro biological evaluation of an array of potent retinoid-X-receptor (RXR) agonists employing 6-(ethyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)nicotinic acid (NEt-TMN), and recently reported NEt-TMN analogs, as a case study. These methods have been extensively applied beyond the present case study to generate several analogs of other potent RXR agonists (rexinoids), particularly the RXR agonist known as bexarotene (Bex), a Food and Drug Administration (FDA) approved drug for cutaneous T-cell lymphoma that is also often prescribed, off-label, for breast, lung, and other human cancers. Common side effects with Bex treatment include hypertriglyceridemia and hypothyroidism, because of off-target activation or inhibition of other nuclear receptor pathways impacted by RXR. Because rexinoids are often selective for RXR, versus the retinoic-acid-receptor (RAR), cutaneous toxicity is often avoided as a side effect for rexinoid treatment. Several other potent RXR agonists, and their analogs, have been reported in the literature and rigorously evaluated (often in comparison to Bex) as potential cancer therapeutics with unique activity and side-effect profiles. Some of the more prominent examples include LGD100268, CD3254, and 9-cis-UAB30, to name only a few. Hence, the methods described herein are more widely applicable to a diverse array of RXR agonists.In terms of design, the structure-activity relationship (SAR) study is usually performed by modifying three distinct areas of the rexinoid base structure, either of the nonpolar or polar sides of the rexinoid and/or the linkage that joins them. For the synthesis of the modified base-structure analogs, often identical synthetic strategies used to access the base-structure are applied; however, reasonable alternative synthetic routes may need to be explored if the modified analog intermediates encounter bottlenecks where yields are negligible for a given step in the base-structure route. In fact, this particular problem was encountered and successfully resolved in our case study for generating an array of NEt-TMN analogs.

This chapter outlines the materials, methods, and procedures for the in vitro biological evaluation of retinoid-X-receptor (RXR) agonists including 6-(ethyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)nicotinic acid (NEt-TMN), as well as several NEt-TMN analog compounds recently reported by our group. These methods have general applicability beyond this NEt-TMN case study, and can be employed to characterize and biologically evaluate other putative RXR agonists (rexinoids), and benchmarked against perhaps the most common rexinoid known as bexarotene (Bex), a drug awarded FDA approval for the treatment of cutaneous T-cell lymphoma in 1999 but that is also prescribed for non-small cell lung cancer and continues to be explored in multiple human cancer types. The side-effect profile of Bex treatment includes hypothyroidism and hypertriglyceridemia arising from the inhibition or activation of additional nuclear receptors that partner with RXR. Because rexinoids often exhibit selectivity for RXR activation, versus activating the retinoic-acid-receptor (RAR), rexinoid treatment avoids the cutaneous toxicity commonly associated as a side effect with retinoids. There are many examples of other potent rexinoids, where biological evaluation has contributed useful insight into qSAR studies on these compounds, often also benchmarked to Bex, as potential treatments for cancer. Because of differential pleiotropy in other pathways, even closely related rexinoids display unique side-effect and activity profiles. Notable examples of potent rexinoids in addition to Bex and NEt-TMN include CD3254, LGD100268, and 9-cis-UAB30. Indeed, the methods described herein to evaluate NEt-TMN and analogous rexinoids are generally applicable to a wider variety of potent, moderate, and even weak RXR ligands.In terms of in vitro biological evaluation, methods for a rapid and preliminary assessment of rexinoid activity are described by employing a biologically relevant, RXR-responsive element (RXRE)-mediated transcription assay in mammalian cells. In addition, a second, more sensitive assay is also detailed that utilizes activation of RXR-RXR homodimers in the context of a mammalian two-hybrid (M2H) luciferase assay. Methods for applying the M2H assay at different rexinoid concentrations are further described for the determination of EC50 values for rexinoids from dose-response curves.

Nuclear 9-cis retinoic acid receptors (retinoid X receptors, RXR) are promiscuous dimerization partners for a number of nuclear receptors. In the present study, we established a novel in vitro method for quantitative determination of the nuclear retinoid X receptors in rat liver. One type of high affinity and limited capacity RXR specific binding sites with the Ka value ranging from 1.011 to 1.727×10(9)l/mol and the Bmax value ranging from 0.346 to 0.567pmol/mg, was demonstrated. Maximal 9-cis retinoic acid (9cRA) specific binding to nuclear retinoid X receptors was achieved at 20°C, and the optimal incubation time for the 9cRA-RXR complex formation was 120min. From a number of endocrine disruptors, tributyltins and triphenyltins are known as RXR ligands. Our data confirmed the property of tributyltin chloride or triphenyltin chloride to bind to a high affinity and limited capacity RXR binding sites. Described optimal conditions for ligand binding to RXR molecules enabled us to calculate maximal binding capacity (Bmax) and affinity (Ka) values. This study provides an original RXR radioligand binding assay that can be employed for investigation of novel RXR ligands that comprise both drugs and endocrine disruptors.