So far, about 130 disinfection by-products (DBPs) and several DBPs-groups have had their potential endocrine-disrupting effects tested on some endocrine endpoints. However, it is still not clear which specific DBPs, DBPs-groups/subgroups may be the most toxic substances or groups/subgroups for any given endocrine endpoint. In this study, we attempt to address this issue. First, a list of relevant DBPs was updated, and 1187 DBPs belonging to 4 main-groups (aliphatic, aromatic, alicyclic, heterocyclic) and 84 subgroups were described. Then, the high-priority endocrine endpoints, DBPs-groups/subgroups, and specific DBPs were determined from 18 endpoints, 4 main-groups, 84 subgroups, and 1187 specific DBPs by a virtual-screening method. The results demonstrate that most of DBPs could not disturb the endocrine endpoints in question because the proportion of active compounds associated with the endocrine endpoints ranged from 0 (human thyroid receptor beta) to 32% (human transthyretin (hTTR)). All the endpoints with a proportion of active compounds greater than 10% belonged to the thyroid system, highlighting that the potential disrupting effects of DBPs on the thyroid system should be given more attention. The aromatic and alicyclic DBPs may have higher priority than that of aliphatic and heterocyclic DBPs by considering the activity rate and potential for disrupting effects. There were 2 (halophenols and estrogen DBPs), 12, and 24 subgroups that belonged to high, moderate, and low priority classes, respectively. For individual DBPs, there were 23 (2%), 193 (16%), and 971 (82%) DBPs belonging to the high, moderate, and low priority groups, respectively. Lastly, the hTTR binding affinity of 4 DBPs was determined by an in vitro assay and all the tested DBPs exhibited dose-dependent binding potency with hTTR, which was consistent with the predicted result. Thus, more efforts should be performed to reveal the potential endocrine disruption of those high research-priority main-groups, subgroups, and individual DBPs.

Perfluorooctanoic acid (PFOA), a synthetic perfluorinated eight-carbon organic chemical, induces hepatotoxicity in rodents, indicated increased liver weight, hepatocellular hypertrophy, necrosis, and peroxisome proliferation. Epidemiological studies have demonstrated the association between serum PFOA levels and various adverse effects. In this study, we investigated the gene expression profiles of human HepaRG cells exposed to 10 and 100 μM PFOA for 24 h. Treatment with 10 and 100 μM PFOA significantly modulated the expression of 190 and 996 genes, respectively. Genes upregulated or downregulated by 100 µM PFOA included peroxisome proliferator-activated receptor (PPAR) signaling genes related to lipid metabolism, adipocyte differentiation, and gluconeogenesis. Moreover, we identified the \"Nuclear receptors-meta pathways\" following the activation of other nuclear receptors: constitutive androstane receptor (CAR), pregnane X receptor (PXR) and farnesoid X receptor (FXR), as well as the transcription factor nuclear factor E2-related factor 2 (Nrf2). The expression levels of some target genes (CYP4A11, CYP2B6, CYP3A4, CYP7A1, and GPX2) of these nuclear receptors and Nrf2 were confirmed using quantitative reverse transcription polymerase chain reaction. Next, we performed transactivation assays using COS-7 and HEK293 cells to investigate whether these signaling-pathways were activated by the direct effects of PFOA on human PPARα, CAR, PXR, FXR and Nrf2. PFOA concentration-dependently activated PPARα, but not CAR, PXR, FXR, or Nrf2. Taken together, these results suggest that PFOA affects the hepatic transcriptomic responses of HepaRG cells through the direct activation of PPARα and indirect activation of CAR, PXR, FXR, and Nrf2. Our finding indicates that PPARα activation in the \"Nuclear receptors-meta pathways\" functions as a molecular initiating event for PFOA, and indirect activation of alternative nuclear receptors and Nrf2 also induce important molecular mechanisms in PFOA-induced human hepatotoxicity.

Few in vitro models are used to study mammary epithelial cells (MECs), and most of these do not express the estrogen receptor α (ERα). Primary MECs can be used to overcome this issue, but methods to purify these cells generally require flow cytometry and fluorescence-activated cell sorting (FACS), which require specialized instruments and expertise. Herein, we present in detail a FACS-free protocol for purification and primary culture of mouse MECs. These MECs remain differentiated for up to six days with >85% luminal epithelial cells in two-dimensional culture. When seeded in Matrigel, they form organoids that recapitulate the mammary gland\'s morphology in vivo by developing lumens, contractile cells, and lobular structures. MECs express a functional ERα signaling pathway in both two- and three-dimensional cell culture, as shown at the mRNA and protein levels and by the phenotypic characterization. Extracellular metabolic flux analysis showed that estrogens induce a metabolic switch favoring aerobic glycolysis over mitochondrial respiration in MECs grown in two-dimensions, a phenomenon known as the Warburg effect. We also performed mass spectrometry (MS)-based metabolomics in organoids. Estrogens altered the levels of metabolites from various pathways, including aerobic glycolysis, citric acid cycle, urea cycle, and amino acid metabolism, demonstrating that ERα reprograms cell metabolism in mammary organoids. Overall, we have optimized mouse MEC isolation and purification for two- and three-dimensional cultures. This model represents a valuable tool to study how estrogens modulate mammary gland biology, and particularly how these hormones reprogram metabolism during lactation and breast carcinogenesis.

Polychlorinated dibenzo-p-dioxins (PCDDs) are hypothesized to exert their toxic effects in wildlife and humans via endocrine disruption. However, very scanty information is available on the underlying molecular interactions that trigger this disruption. In this study, molecular docking simulation was used to predict the susceptibility of 12 nuclear receptors to disruption via PCDD bindings. Findings revealed that androgen (AR and AR an), estrogen (ER α and ER β), glucocorticoid (GR) and thyroid hormone (TR α and TR β) receptors are the most probable protein targets that bind to PCDDs. Further molecular docking analyses showed that PCDD molecules mimic the modes of interaction observed for the co-crystallized ligands of the affected receptors, resulting in the formation of ligand-receptor complexes that were stabilized through electrostatic, van der Waals, pi-effect and hydrophobic interactions with 18, 17, 17, 16, 18, eight and four amino acid residues in the active sites of AR, AR an, ER α, ER β, GR, TR α and TR β respectively. The commonalities of these interacting amino acid residues with those utilized by dihydrotestosterone in AR, bicalutamide in AR an, 17β-estradiol in ER α, 17β-estradiol in ER β, cortisol in GR, thyromimetic GC-1 in TR α and thyromimetic GC-1 in TR β are 86%, 74%, 94%, 80%, 82%, 50% and 43% respectively. The results obtained in this study provide supporting evidence that PCDD molecules may interfere with the endocrine system via binding interactions with some vital amino acid residues in the binding pockets of AR, ERs, GRs and TRs.

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.

Time-resolved fluorescence resonance energy transfer (TR-FRET) protein-protein interaction assays, especially in the format of receptor coregulator (coactivator and corepressor) recruitment/repression assays, have been widely used in nuclear receptor research to characterize the modes of action, efficacies, and binding affinities of ligands (including their properties as agonists, antagonists, and inverse agonists). However, there has been only limited progress in using this assay format for pregnane X receptor (PXR). In this chapter, we discuss TR-FRET protein-protein interaction assays and focus on a novel PXR TR-FRET coactivator interaction assay that we have developed based on a PXR coactivator cocrystal study. This new PXR TR-FRET coactivator interaction assay can characterize the binding affinities of PXR ligands and also differentiate antagonists from agonists. This assay is very robust, with the signal remaining stable over a long incubation time (up to 300min has been tested). It can tolerate high concentrations of DMSO (up to 5%) and has a high signal-to-noise ratio (six under typical assay conditions). This newly developed PXR TR-FRET coactivator interaction assay has potential application in high-throughput screening to identify and characterize novel PXR agonists and antagonists.