Binding profiles of human and mouse complement component 8γ to trisubstituted organometallic compounds.

Complement component 8gamma (C8γ), a member of the lipocalin protein family, is suggested to act as a carrier protein for various chemicals. Although C8γ has been identified in both humans and rodents for some time, our understanding of the species differences in its chemical binding properties remains limited. In the present study, with the aim to elucidate the potential role of C8γ as a carrier protein in both humans and mice, we conducted a radioligand binding assay to examine the chemical binding properties of human C8γ (hC8γ) and mouse C8γ (mC8γ). Scatchard analysis revealed that [14C]TPT bound to hC8γ with an equilibrium dissociation constant (Kd) of 64.2 ± 32.4 nM, comparable to that of [14C]TPT to mC8γ. Competitive ligand-binding assays demonstrated binding of TPT and TBT to hC8γ, while diphenyltin, dibutyltin, monophenyltin, monobutyltin, and tetrabutyltin did not exhibit binding. These results suggest that for effective binding to C8γ, chemicals must possess substituents of appropriate bulkiness. Further analyses with other group 14 compounds with triphenyl substituents revealed that a central metal atom, rather than a central non-metal or semi-metal atom, is crucial for specific binding to both hC8γ and mC8γ. Overall our findings imply that C8γ may play a role in the physiological or toxicological actions of group 14 metal compounds with tributyl or triphenyl substituents by binding to these chemicals in both humans and mice.

Determination of a Serum 25-Hydroxyvitamin D Reference Ranges in Japanese Adults Using Fully Automated Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Despite an increasing interest in vitamin D status, a reference range of the nutrient has not been fully established. This is partly due to a paucity of standardized measuring systems with high throughput. In addition, the range may vary by populations and may change with modernization of lifestyles. OBJECTIVES: This study aims to calculate the current reference concentration of 25-hydroxyvitamin D (25(OH)D) among healthy people living in an urban area in Japan. METHODS: A newly developed fully automated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) system was used to measure serum 25(OH)D concentrations. Reproducibility was assessed by measuring standardized samples. Accuracy was validated by comparing with commercially available immunoassays. Then, mass screening was conducted targeting participants who received medical checkups in Tokyo from April 2019 to March 2020, and the reference ranges were calculated. RESULTS: The coefficients of variations of interoperator and interday reproducibility were 4.1%-8.5% and 3.7%-8.0% for 25-hydroxyvitamin D2 (25(OH)D2) and 4.7%-7.0% and 4.0%-6.9% for 25-hydroxyvitamine D3, respectively. The measured total 25(OH)D concentrations correlated well with those measured by immunoassays. In total, 5518 participants were measured for 25(OH)D concentrations, among whom 98% showed inadequate concentrations (<30 ng/mL). The reference ranges of total 25(OH)D for female, male, and total participants were 7-30 ng/mL, 5-27 ng/mL, and 6-29 ng/mL, respectively. After excluding those with abnormal renal and liver function, the range was 6-30 ng/mL. CONCLUSIONS: The high prevalence of vitamin D insufficiency among seemingly healthy population may be attributed to lifestyle characteristics of people living in urban areas of Japan, including spending less time outdoors and lower intake of traditional foods. Longitudinal follow-up and mass screenings targeting different population will help elucidate reasons for discrepancies between official guidelines and the observed concentrations, to which the well-validated measurement system is essential.

Novel, highly sensitive, in vivo screening method detects estrogenic activity at low doses of bisphenol A.

Low doses of bisphenol A (BPA), a typical endocrine-disrupting chemical (EDC), have been reported to exhibit estrogenic action in animals; however, the effects have not been fully clarified because of their non-reproducibility. Here, we developed a novel, short-term screening test for estrogen-like chemicals using in vivo bioluminescence imaging of estrogen-responsive reporter (E-Rep) mice. Comparative studies using 17α-ethinylestradiol and selective estrogen receptor modulators demonstrated that the method provides higher detection sensitivity and requires less time than the uterotrophic bioassay, a well-established, in vivo screening method for estrogen-like chemicals. Our method could detect the estrogenic effects of BPA at doses below tolerable daily intakes, whereas the uterotrophic bioassay could not. Our results indicated that in vivo bioluminescence imaging using E-Rep mice was extremely useful for screening estrogenic chemicals and detecting estrogenic effects at low doses of EDCs, including BPA. Our method should help resolve the controversy about low-dose effects of EDCs.

Cadmium inhibits differentiation of human trophoblast stem cells into extravillous trophoblasts and disrupts epigenetic changes within the promoter region of the HLA-G gene.

Cadmium (Cd) is a toxic heavy metal widely distributed in the environment. Maternal whole-blood Cd levels during pregnancy are positively associated with the risk of early preterm birth. We hypothesized that Cd inhibits trophoblast differentiation, resulting in the development of hypertensive disorders of pregnancy and a high risk of early preterm birth. Using the CT27 human trophoblast stem cell line, we found that exposing these cells to 0.1-0.4 µM Cd inhibited their differentiation into extravillous cytotrophoblasts (EVTs). Supporting this finding, we found that expression of the metal-binding protein metallothionein, which suppresses the toxicity of Cd, is low in EVTs. We also found that Cd exposure changes the methylation status of the promoter region of the HLA-G gene, which is specifically expressed in EVTs. Together, these results suggest that Cd inhibits placental formation by suppressing trophoblast differentiation into EVTs. This suppression may underlie the increased risk of gestational hypertension in women with high whole-blood Cd levels.

Neuronal differentiation reporter mice as a new methodology for detecting in vivo developmental neurotoxicity.

Current in vivo developmental neurotoxicity (DNT) tests are not performed routinely for chemical risk assessment because they are time and resource intensive and require many animals. Therefore, new methodologies are required that can detect and evaluate the DNT potential of chemicals in a more simple, quantitative, and objective manner. Toward this end, we generated transgenic mice expressing reporter genes (luciferase and lacZ) under the control of the rat synapsin 1 promoter (Syn-Rep mice) and evaluated their usefulness as a DNT detection tool. Brain luciferase expression levels in Syn-Rep mice increased dramatically from just before to after birth, peaked early in the postnatal period, subsequently decreased sharply, and then remained low after weaning. This pattern is analogous to the generally recognized temporal changes in synapse numbers in the developing mammal brain. To evaluate further the responsiveness of Syn-Rep mice during DNT induction, we administered valproic acid (VPA), a reference DNT-inducing chemical, to pregnant mice and evaluated its effect on reporter gene expression in the developing brains of Syn-Rep pups. In vivo luminescence in the brains of VPA-exposed pups was significantly lower than in controls from postnatal days 4 to 13. Moreover, luciferase activity in the prefrontal cortexes of 8-week-old VPA-exposed offspring was significantly lower than in controls, reflecting the reduced number of neurons in the prefrontal cortex. These results suggest that the Syn-Rep mice are potentially useful tools for streamlined detection of chemical-induced DNT in the developing mammalian brain.

Exposure to the Organophosphate Pesticide Fenitrothion Directly Induced Defects in Mouse Embryonic External Genitalia.

Many industrial chemicals have been reported as antiandrogenic substances. Exposure to these substances represents a potential risk to human health, particularly to the development of reproductive organs such as embryonic external genitalia (eExG). Currently, there is a need for more assay systems that can elucidate the toxicological actions and mechanisms of endocrine-disrupting chemicals. In this study, we show that the eExG slice culture assay is useful for the evaluation of the differing modes of action of endocrine-disrupting chemicals on urethra formation. We assessed the possible endocrine-disrupting activity of 3 chemicals with reported antiandrogenic function, diazinon, dibutyl phthalate, and fenitrothion (FNT) on eExG slices. Exposure to FNT, but not diazinon and dibutyl phthalate, induced defects of androgen-induced urethral masculinization and reduced expression of the androgen-target gene Mafb. Live imaging analyses showed that FNT treatment inhibited androgen-dependent MAFB induction within 12 h. Furthermore, FNT-treated tissue slices showed reduced expression of the androgen receptor. These results indicate that FNT disrupts androgen signaling by reduction of androgen receptor expression during androgen-induced eExG masculinization. This study thus highlights the importance of animal models, which allow for the effective assessment of tissue-specific endocrine-disrupting activity to further reveal the etiology of chemical-induced congenital anomalies.

Evaluation of the Estrogenic Action Potential of Royal Jelly by Genomic Signaling Pathway in Vitro and in Vivo.

Royal jelly (RJ) has beneficial effects on human health, and some of these effects are reported to be the result of its estrogenic activity; however, chemicals with estrogenic activities may disrupt physiological estrogen signaling leading to adverse effects on human health. Thus, clarification of the mode of action of RJ is needed. Here, we investigated whether the estrogen-like actions of RJ are induced via estrogen receptors (ERs)-mediated genomic actions by using an in vitro reporter assay in human choriocarcinoma JEG3 cells and an estrogen-responsive reporter (E-Rep) mouse line that can be used to sensitively detect transactivation of ERs in multiple organs simultaneously. In the in vitro reporter assay, ERs-dependent transcriptional activity was significantly increased by 17ß-estradiol (E2) treatment at concentrations of 1 nM and above, confirming that the assay was highly responsive to estrogen; however, RJ did not exhibit any agonist activity via either the α or ß form of ER. Similarly, in E-Rep mice, E2 showed significant ERs-dependent genomic action in 17 tissue types including uterus and mammary gland, whereas RJ did not. Thus, unlike endocrine-disrupting chemicals, the estrogen-like activity of RJ is unlikely to be due to genomic actions via ERs.

Cadmium inhibits forskolin-induced differentiation of human placental BeWo cells.

Cadmium (Cd) is an environmental pollutant. Blood Cd levels in pregnant women have been associated with premature births, infant birth size, placenta previa, and placenta accreta. There have been concerns on the reproductive developmental toxicity of Cd. The choriocarcinoma cell line BeWo, a cellular in vitro model for studying syncytial fusion, has been widely used to study the reproductive and developmental toxic effects of pollutants. Here, we examine the inhibitory effect of Cd against forskolin (FSK)-induced BeWo differentiation. Results showed that Cd exposure inhibited the FSK-induced expression of syncytiotrophoblast-related genes LGALS13, ERVFRD1, SDC1, and CGB3. Inhibition of LGALS13 expression was due to the inhibition of the PKA pathway, whereas the inhibition of the other three genes could be due to the inhibition of the other pathways. These findings could help clarify the reproductive and developmental toxicity of Cd.

Discovery of contaminants with antagonistic activity against retinoic acid receptor in house dust.

Retinoic acid receptors (RARs) control reproduction and development in vertebrates, but little attention has been paid to anthropogenic chemicals exhibiting RAR agoniztic/antagonistic activity. Here we applied a His-RARα pull-down assay combined with high-resolution mass spectrometry to identify chemicals with RARα activity in house dust. After screening, a total of 540 peaks were retained as potential RARα ligands. The mass spectra of 14 chemicals matched with those in the database, of which triphenyl phosphate, galaxolidone, di(2-ethylhexyl) phthalate (DEHP), tris(2-ethylhexyl) phosphate (TEHP), and tris(2-butoxyethyl) phosphate were confirmed by their standards. While one chemical in the sample matched with monophenyl phosphate in the MS/MS database, its retention time was much higher than that of monophenyl phosphate standard, suggesting that it may be an in-source fragment. Its parent ion was finally identified to be m/z 399.2663 using a similarity analysis among chromatographic peaks of hundreds of ions at the same retention time in MS1 spectrum, and bis(2-ethylhexyl) phenyl phosphate (BEHPP) was identified. BEHPP, DEHP, and TEHP were for the first time identified to be RARα antagonists with IC50 values of 6556, 6600, and 2538 nM, respectively. This study improved structural annotation and filled the knowledge gap regarding widespread environmental contaminants with RAR antagonistic activity.

Evaluation of the Skin-Sensitizing Potential of Brazilian Green Propolis.

Propolis is a resinous mixture produced by bees from their secretions and plant material, so its composition varies depending on its botanical origin. Propolis has several beneficial bioactivities, but its skin sensitization properties have long been suspected. Nevertheless, the skin sensitization potency of Brazilian green propolis (BGP) has not been scientifically evaluated. Here, we used scientifically reliable tests to evaluate it. In vitro antigenicity test based on the human cell line activation test (OECD TG 442E) was performed by measuring the expression of CD54 and CD86, which are indicators of the antigenicity of test substances, on THP-1 and DC2.4 cells. BGP did not affect the expression of either marker on THP-1 cells, but upregulated the expression of CD86 on DC2.4 cells, suggesting that BGP may be a skin sensitizer. Then, we performed local lymph node assay (LLNA, OECD TG 429) as a definitive in vivo test. LLNA showed that 1.70% BGP primed skin sensitization and is a "moderate sensitizer". Our results indicate scientific proof of the validity of arbitrary concentrations (1-2%), which have been used empirically, and provide the first scientific information on the safe use of BGP.

Triphenyl phosphate delayed pubertal timing and induced decline of ovarian reserve in mice as an estrogen receptor antagonist.

Although concerns have been raised about the adverse effects of triphenyl phosphate (TPhP) on female fertility, its risk to ovarian functioning remains unknown. In this study, female C57BL/6 mice at postnatal day 21 were exposed on a daily basis to TPhP dose of 2, 10, and 50 mg/kg for 40 days. A significant delay in pubertal timing was observed in the mice exposed to 50 mg/kg of TPhP. An estrogen-responsive reporter transgenic mice assay demonstrated that TPhP significantly downregulated the estrogen receptor (ER) signaling by 45.1% in the whole body in the 50 mg/kg group, and by 14.7-43.7% in the uterus for all exposure groups compared with the control. This strong antagonistic activity of TPhP toward ER explained the delay in pubertal timing. A significant reduction in the number of follicles in all stages was observed in mice after being exposed to TPhP for 40 days at concentrations of 10 and 50 mg/kg, resulting in a decline of the ovarian reserve. The elevation of the follicle-stimulating hormone concentration may have contributed to this phenomenon, as controlled by the antagonistic activity of TPhP toward ER in the brain. The toxic effects of TPhP on ovarian functioning highlight this chemical as a potential risk factor for female fertility.

Alginate-coated activated charcoal enhances fecal excretion of 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice, with fewer side effects than uncoated one.

Activated charcoal (AC) is a potential candidate antidote against dioxins. However, it is difficult to take AC as a supplement on a daily basis, because its long-term ingestion causes side effects such as constipation and deficiency of fat-soluble essential nutrients and hypocholesterolemia. Alginate-coated AC, termed Health Carbon (HC), was developed to decrease the side effects of AC, but its pharmacological effects, including side effects, remains unclear. Here, we show that HC enhanced fecal excretion of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and decreased some side effects of unmodified AC, such as hypocholesterolemia, in male mice. Basal diet mixed with HC or unmodified AC at various concentrations was fed to mice for 16 days following a single intraperitoneal administration of [3H]TCDD. Both HC and unmodified AC at 3% or more significantly increased fecal excretion of [3H]TCDD in comparison with the control basal diet. Consistent with this, [3H]TCDD radioactivity in the liver-a major TCDD storage organ-was markedly decreased by HC at concentrations of 3% and 10%. In an examination of potential side effects, unmodified AC at 10% or more caused significant body weight reduction and at 20% caused significant hypocholesterolemia. In contrast, HC caused weight gain reduction only at a concentration of 20%, and there was no evidence of hypocholesterolemia at any dietary HC concentration. HC not only retains the ability of AC to enhance fecal excretion of TCDD but also reduces some of the side effects of AC.

An ancestral nuclear receptor couple, PPAR-RXR, is exploited by organotins.

Environmental chemicals have been reported to greatly disturb the endocrine and metabolic systems of multiple animal species. A recent example involves the exploitation of the nuclear receptor (NR) heterodimeric pair composed by PPAR/RXR (peroxisome proliferator-activated receptor/retinoid X receptor), which shows lipid perturbation in mammalian species. While gene orthologues of both of these receptors have been described outside vertebrates, no functional characterization of PPAR has been carried in protostome lineages. We provide the first functional analysis of PPAR in Patella sp. (Mollusca), using model obesogens such as tributyltin (TBT), triphenyltin (TPT), and proposed natural ligands (fatty acid molecules). To gain further insights, we used site-directed mutagenesis to PPAR and replaced the tyrosine 277 by a cysteine (the human homologous amino acid and TBT anchor residue) and an alanine. Additionally, we explored the alterations in the fatty acid profiles after an exposure to the model obesogen TBT, in vivo. Our results show that TBT and TPT behave as an antagonist of Patella sp. PPAR/RXR and that the tyrosine 277 is important, but not essential in the response to TBT. Overall, these results suggest a relation between the response of the mollusc PPAR-RXR to TBT and the lipid profile alterations reported at environmentally relevant concentrations. Our findings highlight the importance of comparative analysis between protostome and deuterostome lineages to decipher the differential impact of environmental chemicals.

Maternal Transfer of 2-Ethylhexyl Diphenyl Phosphate Leads to Developmental Toxicity Possibly by Blocking the Retinoic Acid Receptor and Retinoic X Receptor in Japanese Medaka (Oryzias latipes).

2-Ethylhexyl diphenyl phosphate (EHDPP) has been detected in wild fish with high concentrations, which may pose a risk in the embryo development considering its potential maternal transfer. In this study, EHDPP was demonstrated to elicit antagonistic activity to medaka retinoic acid receptor (mRAR) and retinoic X receptor (mRXR) with 50% inhibitory concentration of 18 and 36 µM, respectively. After adult female medaka were exposed to EHDPP at 156, 405, and 1161 ng/L for 35 days, the embryonic EHDPP concentrations (364-4824 ng/g lipid weight (lw)) were higher than those in the maternal tissues (15.0-4166 ng/g lw), showing notable maternal transfer. The embryonic concentration of EHDPP decreased limitedly during 1-2 day post-fertilization (dpf, the main developmental window of eye) but then decreased sharply after 2 dpf. The transcript abundance of cyp26a1 was inhibited and subsequent increasing embryonic all-trans RA level was observed in embryos, showing RAR/RXR antagonistic activity. These results may specifically contribute to the increased eye deformity incidences in all exposure groups (up to 8.0%; 51/637) relative to the control (1.0%, 7/733). The response behavior of the larvae to light stimulation was impaired in a dose-dependent manner, demonstrating a vision disorder. Because such developmental toxicities were observed at the environmental level, EHDPP may pose a threat to the survival of wild larvae and therefore a population risk for wild fish.

Fake metabolomics chromatogram generation for facilitating deep learning of peak-picking neural networks.

Finding peaks in chromatograms and determining their start and end points (peak picking) is a core task in chromatography based biotechnology. Construction of peak-picking neural networks by deep learning was, however, hampered from the preparation of exact peak-picked or "labeled" chromatograms since the exact start and end points were often unclear in overlapping peaks in real chromatograms. We present a design of a fake chromatogram generator, along with a method for deep learning of peak-picking neural networks. Fake chromatograms were generated by generation of fake peaks, random sampling of peak positions from feature distributions, and merging with real blank sample chromatograms. Information on the exact start and end points, as labeled on the fake chromatograms, were effective for training and evaluating peak-picking neural networks. The peak-picking neural networks constructed herein outperformed conventional peak-picking software and showed comparable performance with that of experienced operators for processing the widely targeted metabolome data. Results of this study indicate that generation of fake chromatograms would be crucial for developing peak-picking neural networks and a key technology for further improvement of peak picking neural networks.

Tri-substituted organotin compounds, but not retinoic acid, are potent ligands of complement component 8 γ.

Complement component 8 γ (C8γ) is a subunit of complement protein 8 (C8), which itself is a subunit of the complement cytolytic membrane attack complex. However, C8γ is also suggested to be a carrier protein for the general clearance of endogenous and exogenous compounds because it belongs to the lipocalin family of small secreted proteins that have the common ability to bind small hydrophobic ligands. Although retinoic acid, a metabolite of vitamin A, has been suggested as a potential ligand of C8γ, it remains unclear which other substances are able to bind to C8γ as ligands. Here, we evaluated the binding affinity of several organotin compounds that are ligands of a receptor of retinoic acid, retinoid X receptor, by using radioligand binding assays. The amount of [14C]triphenyltin (TPT), a tri-substituted organotin, that bound to purified recombinant C8γ was increased with increasing protein concentration, whereas that of [3H]all-trans retinoic acid and [3H]9-cis retinoic acid was unchanged. Scatchard analysis revealed that [14C]TPT bound to C8γ with an equilibrium dissociation constant (Kd) of 56.2 ± 16.2 nM. Non-radiolabeled tributyltin (TBT), another tri-substituted organotin, blocked the binding of [14C]TPT to C8γ in a competitive manner, but non-radiolabeled mono- or di-substituted organotin compounds did not. Together, our present observations indicate that TBT and TPT, but not retinoic acid or mono- or di-substituted organotin compounds, are potent ligands of C8γ, suggesting that C8γ may be involved in the toxicities of these organotin compounds.

CpG Site-Specific Regulation of Metallothionein-1 Gene Expression.

Metal-binding inducible proteins called metallothioneins (MTs) protect cells from heavy-metal toxicity. Their transcription is regulated by metal response element (MRE)-binding transcription factor-1 (MTF1), which is strongly recruited to MREs in the MT promoters, in response to Zn and Cd. Mouse Mt1 gene promoter contains 5 MREs (a-e), and MTF1 has the highest affinity to MREd. Epigenetic changes like DNA methylation might affect transcription and, therefore, the cytoprotective function of MT genes. To reveal the CpG site(s) critical for Mt1 transcription, we analyzed the methylation status of CpG dinucleotides in the Mt1 gene promoter through bisulfite sequencing in P1798 mouse lymphosarcoma cells, with high or low MT expression. We found demethylated CpG sites near MREd and MREe, in cells with high expression. Next, we compared Mt1 gene-promoter-driven Lucia luciferase gene expression in unmethylated and methylated reporter vectors. To clarify the effect of complete and partial CpG methylation, we used M.SssI (CG→5mCG) and HhaI (GCGC→G5mCGC)-methylated reporter vectors. Point mutation analysis revealed that methylation of a CpG site near MREd and MREe strongly inhibited Mt1 gene expression. Our results suggest that the methylation status of this site is important for the regulation of Mt1 gene expression.

In vivo profiling of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced estrogenic/anti-estrogenic effects in female estrogen-responsive reporter transgenic mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), commonly referred to simply as "dioxin", is a persistent environmental pollutant. Because of its high environmental persistence and biological accumulation, humans and animals are often exposed to TCDD. Therefore, the harmful effects on humans and animals is a major concern. Although studies have elucidated the adverse estrogenic and anti-estrogenic effects of TCDD, it is unclear in which tissues TCDD exerts these effects in vivo. To investigate the estrogen-related effects of TCDD in various tissues, we generated an improved estrogen-responsive reporter transgenic mouse in which the luciferase gene luc2 is expressed in response to estrogenic signals. Using these mice, we clarified that TCDD inhibits estrogenic signaling in liver and kidney but enhances estrogenic signaling in the pituitary gland in the same individual. Expression of aryl hydrocarbon receptor, aryl hydrocarbon receptor nuclear translocator, and estrogen receptor alpha mRNA was detected in liver, kidney, and pituitary gland, suggesting that the effects of TCDD on estrogenic signaling in these organs is independent of the expression pattern of these receptors. Thus, our results indicate that TCDD exerts both estrogenic and anti-estrogenic tissue-specific effects within the same individual.

An Orthologue of the Retinoic Acid Receptor (RAR) Is Present in the Ecdysozoa Phylum Priapulida.

Signalling molecules and their cognate receptors are central components of the Metazoa endocrine system. Defining their presence or absence in extant animal lineages is critical to accurately devise evolutionary patterns, physiological shifts and the impact of endocrine disrupting chemicals. Here, we address the evolution of retinoic acid (RA) signalling in the Priapulida worm, Priapuluscaudatus Lamarck, 1816, an Ecdysozoa. RA signalling has been shown to be central to chordate endocrine homeostasis, participating in multiple developmental and physiological processes. Priapulids, with their slow rate of molecular evolution and phylogenetic position, represent a key taxon to investigate the early phases of Ecdysozoa evolution. By exploring a draft genome assembly, we show, by means of phylogenetics and functional assays, that an orthologue of the nuclear receptor retinoic acid receptor (RAR) subfamily, a central mediator of RA signalling, is present in Ecdysozoa, contrary to previous perception. We further demonstrate that the Priapulida RAR displays low-affinity for retinoids (similar to annelids), and is not responsive to common endocrine disruptors acting via RAR. Our findings provide a timeline for RA signalling evolution in the Bilateria and give support to the hypothesis that the increase in RA affinity towards RAR is a late acquisition in the evolution of the Metazoa.

Screening of House Dust from Chinese Homes for Chemicals with Liver X Receptors Binding Activities and Characterization of Atherosclerotic Activity Using an in Vitro Macrophage Cell Line and ApoE-/- Mice.

BACKGROUND: Atherosclerotic cardiovascular disease has become the leading cause of death worldwide, and environmental pollutants are increasingly recognized as risk factors for atherosclerosis. Liver X receptors (LXRs) play a central role in atherosclerosis; however, LXR activity of organic pollutants and associated potential risk of atherosclerosis have not yet been characterized. OBJECTIVES: This study aimed to explore whether LXR-antagonistic chemicals are present in indoor house dust and, if so, to characterize this activity in relation to changes in macrophages in vitro and cardiovascular disease indicators in vivo in an atherosclerosis ApoE-/- mouse model. METHODS: We used a His-LXRα-pull-down assay and a nontarget high-resolution mass spectrometry method to screen house dust collected from Chinese homes for LXRα- and LXRß-antagonist activity. A chemical identified in this manner was assessed for its ability to induce cholesterol efflux and foam cell formation in RAW264.7 macrophages, to down-regulate the expression of two LXR-dependent genes, ABCA1 and ABCG1, and finally to induce atherosclerotic lesions in vivo using an ApoE-/- mouse model. RESULTS: We identified the flame retardants triphenyl phosphate (TPHP) and 2-ethylhexyl diphenyl phosphate (EHDPP) in house dust samples and demonstrated their ability to antagonize LXRs. The potency of TPHP was similar to that of the LXR-antagonist SR9238. TPHP could also inhibit cholesterol efflux and promote foam cell formation in RAW264.7 macrophages and mouse peritoneal macrophages and significantly promoted atherosclerotic lesion formation in the ApoE-/- mouse model. CONCLUSIONS: We found LXR-antagonist chemicals in environmental samples of indoor dust from Chinese homes. One of the chemicals, TPHP, was able to promote the development of atherosclerotic lesions in the ApoE-/- mouse model. These results highlight the need to assess the LXR-antagonist activities of pollutants in future environmental management programs. https://doi.org/10.1289/EHP5039.