Ractopamine, a synthetic β-adrenoreceptor agonist, is used as an animal feed additive to increase food conversion efficiency and accelerate lean mass accretion in farmed animals. The U.S. Food and Drug Administration claimed that ingesting products containing ractopamine residues at legal dosages might not cause short-term harm to human health. However, the effect of ractopamine on chronic inflammatory diseases and atherosclerosis is unclear. Therefore, we investigated the effects of ractopamine on atherosclerosis and its action mechanism in apolipoprotein E-null (apoe⁻/⁻) mice and human endothelial cells (ECs) and macrophages. Daily treatment with ractopamine for four weeks increased the body weight and the weight of brown adipose tissues and gastrocnemius muscles. However, it decreased the weight of white adipose tissues in apoe⁻/⁻ mice. Additionally, ractopamine exacerbated hyperlipidemia and systemic inflammation, deregulated aortic cholesterol metabolism and inflammation, and accelerated atherosclerosis. In ECs, ractopamine treatment induced endothelial dysfunction and increased monocyte adhesion and transmigration across ECs. In macrophages, ractopamine dysregulated cholesterol metabolism by increasing oxidized low-density lipoprotein (oxLDL) internalization and decreasing reverse cholesterol transporters, increasing oxLDL-induced lipid accumulation. Collectively, our findings revealed that ractopamine induces EC dysfunction and deregulated cholesterol metabolism of macrophages, which ultimately accelerates atherosclerosis progression.

Silicosis is a disease mainly caused by pulmonary interstitial fibrosis caused by long-term inhalation of dust with excessively high content of free SiO₂. Transdifferentiation of lung fibroblasts into myofibroblasts is an important cellular basis for silicosis, but the key transcription factors (TFs) involved in this process are still unclear. In order to explore the biological regulation of transcription factor PPARγ/LXRα in silica-induced pulmonary fibrosis, this study explored the molecular mechanism of PPARγ/LXRα involved in regulating transcription factors related to SiO₂-induced lung injury at the cellular level and in animal models. ChIP-qPCR detected that PPARγ directly regulated the transcriptional activity of the LXRα gene promoter, while the PPARγ agonist RSG increased the expression of LXRα. In addition, we demonstrated in the cell model that upregulation of LXRα can inhibit silica-mediated fibroblast transdifferentiation, accompanied by an increase in the expression of SREBF1, PLTP and ABCA1. The results of LXRα silencing experiment matched those of overexpression experiment. These studies explored the role of LXRα in plasticity and phenotypic transformation between lung fibroblasts and myofibroblasts. Therefore, inhibiting or reversing the transdifferentiation of lung fibroblasts to myofibroblasts by intervening PPARγ/LXRα may provide a new therapeutic target for the treatment of silicosis.

We describe the androgen receptor (AR) agonistic/antagonistic effects of 140 veterinary drugs regulated in Republic of Korea, by setting maximum residue limits. It was conducted using two in vitro test guidelines of the Organization for Economic Cooperation and Development (OECD)—the AR-EcoScreen AR transactivation (TA) assay and the 22Rv1/MMTV_GR-KO AR TA assay. These were performed alongside the AR binding affinity assay to confirm whether their AR agonistic/antagonistic effects are based on the binding affinity to AR. Prior to conducting the AR TA assay, the proficiency test was passed the proficiency performance criterion for the AR agonist and AR antagonist assays. Among the veterinary drugs tested, four veterinary drugs (dexamethasone, trenbolone, altrenogest, and nandrolone) and six veterinary drugs (cymiazole, dexamethasone, zeranol, phenothiazine, bromopropylate, and isoeugenol) were determined as AR agonist and AR antagonist, respectively in both in vitro AR TA assays. Zeranol exhibited weak AR agonistic effects with a PC₁₀ value only in the 22Rv1/MMTV_GR-KO AR TA assay. Regarding changing the AR agonistic/antagonistic effects through metabolism, the AR antagonistic activities of zeranol, phenothiazine, and isoeugenol decreased significantly in the presence of phase I + II enzymes.These data indicate that various veterinary drugs could have the potential to disrupt AR-mediated human endocrine system. Furthermore, this is the first report providing information on AR agonistic/antagonistic effects of veterinary drugs using in vitro OECD AR TA assays.

Bisphenol A (BPA) is a well-known for endocrine-disrupting chemical (EDC) and is one of the highest amounts of chemicals produced worldwide. Some countries restrict the use of BPA, which is widely used in the production of a variety products. Considering the toxicity and limitations on use of BPA, efforts are needed to find safer alternatives. Increasingly, bisphenol F (BPF) and bisphenol S (BPS) are alternatives of BPA, which is increasing their exposure levels in various environments. There are many ways to assess whether a chemical is an EDC. Here, we evaluated the endocrine-disrupting risks of the bisphenols by investigating their agonist and antagonist activities with the estrogen (ER), androgen (AR), and aryl hydrocarbon (AhR) receptors. Our results showed that BPA, BPS, and BPF (BPs) have estrogen agonist and androgen antagonist activities and decrease the ERα protein level. Interestingly, a mixture of the BPs had ER and anti-AR activity at lower concentrations than BPs alone. The activation of AhR was not a concentration-dependent effect of BPs, although it was increased significantly. In conclusion, BPs have estrogen agonist and androgen antagonist activities, and the effect of exposure to a BPs mixture differs from that of BPs alone.

Phthalate esters (PAEs), as widely used plasticizers, have been concerned for their possible disruption of estrogen functions via binding to and activating the transcription of estrogen receptors (ERs). Nevertheless, the computational interpretation of the mechanism of ERs activities modulated by PAEs at the molecular level is still insufficient, which hinders the reliable screening of the ERs-active PAEs with high speed and high throughput. To bridge the gap, the in silico simulations considering the effects of coactivators were accomplished to explore the molecular mechanism of action for the purpose of predicting the estrogenic potencies of PAEs. The transcriptional activation functions of human ERα (hERα) modulated by PAEs is predicted via the simulations including binding interaction of PAEs and hERα, conformational changes of PAEs-hERα complexes and recruitment of coactivators. Molecular insight into the diverse estrogen mechanism of action among PAEs with regard to hERα agonists and selective estrogen receptor modulators (SERMs) is provided. Agonist-modulated conformational change of hERα leads to the optimal exposure of its Activation Function 2 (AF-2) surface which, in turn, facilitates the recruitment of coactivators, therefore promoting the transcriptional activation functions of hERα. Conversely, binding interaction of hERα with SERMs among PAEs leads to the conformational change with blocked AF-2 surface, thus preventing the recruitment of coactivators and consequently inhibiting the AF-2 activity. The two-hybrid recombinant yeast is experimentally used for verification. The established in silico evaluation methodology exhibits great promise to speed up the prediction of chemicals which work as hERα agonist or SERMs.

Gut microbiota is of critical importance to host health. Aryl hydrocarbon receptor (AhR) is found to be closely involved in the regulation of gut microbial dynamics. However, it is still not clear how AhR signaling shapes the gut microbiota. In the present study, adult zebrafish were acutely exposed to an AhR antagonist (CH223191), an AhR agonist (polychlorinated biphenyl 126; PCB126) or their combination for 7 d. Overall intestinal health and gut microbial community were temporally monitored (1 d, 3 d and 7 d) and inter-compared among different groups. The results showed that single exposure to PCB126 significantly disrupted the overall health of intestines (i.e., neural signaling, inflammation, epithelial barrier integrity, oxidative stress). However, CH223191 failed to inhibit but enhanced the physiological toxicities of PCB126, implying the involvement of extra mechanisms rather than AhR in the regulation of intestinal physiological activities. Dysbiosis of gut microbiota was also caused by PCB126 over time as a function of sex. It is intriguing that CH223191 successfully abolished the holistic effects of dioxin on gut microbiota, which inferred that growth of gut microbes was directly controlled by AhR activation without the involvement of host feedback modulation. When coming to detailed alterations at certain taxon, both antagonistic and synergistic interactions existed between CH223191 and dioxin, depending on fish sex, exposure duration and bacterial species. Correlation analysis found that gut inflammation was positively associated with pathogenic Legionella bacteria, but was negatively associated with epithelial barrier integrity, suggesting that integral intestinal epithelial barrier can prevent the influx of pathogenic bacteria to induce inflammatory response. Overall, this study has deciphered, for the first time, the direct regulative effects of AhR activity on gut microbiota. Future research is warranted to elucidate the specific mechanisms of AhR action on certain bacterial population.

As a result of human activities, the pollution of metals is becoming ubiquitous in the environment. Among various toxicological mechanisms of action, metals have been considered as endocrine-disrupting chemicals (EDCs) through interference with steroid receptors. However, information regarding the potential endocrine disruption of metals on glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) is especially scarce. In this study, a total of 16 metals were assessed for their GR/MR activities using luciferase reporter gene assay. None of the tested metals exhibited GR or MR agonistic activity, but a total of 7 and 5 candidate metals showed obvious GR and MR antagonistic properties, respectively. All 7 GR antagonistic metals [ BaCl₂, CoCl₂, CuCl₂, Pb(NO₃)₂, LiCl, SnCl₂ and ZnCl₂] inhibited glucocorticoid-responsive gene GILZ expression in J774A.1 cells. Further investigations indicated that the 5 MR antagonistic metals [ CdCl₂, Pb(NO₃)₂, LiCl, MnCl₂ and SnCl₂] antagonized aldosterone-inhibited hepatocellular carcinoma cell proliferation. Among these metals, Pb(NO₃)₂, LiCl, and SnCl₂ showed both anti-glucocorticoid and anti-mineralocorticoid activities. Comprehensive screening and evaluation of GR and MR antagonists and agonists among metals should be considered to better understand the ecological and health risks of metals.

In recent years, some pigments and chemical products have been reported to contain polychlorinated biphenyl (PCB) congeners as unintentional byproducts, and these have also been detected in residential environments from indoor air and house dust. In this study, using in vitro reporter gene assays, we characterized the agonistic and antagonistic activities of a total of 25 PCB congeners contained in pigments (PCB-1 to -16, -20, -35, -40, -52, -56, -77, -101, -126, and -153) against five nuclear hormone receptors, (estrogen receptor (ER) α/β, glucocorticoid receptor (GR), androgen receptor (AR), thyroid hormone receptor (TR) α1) and aryl hydrocarbon receptor (AhR). In the ERα/β assays, 19 and 13 of the 25 PCBs tested showed ERα/β agonistic and/or antagonistic activities, respectively. Relatively potent agonistic activities against ERα/β were found in PCB congeners possessing chlorides at positions 2 and 3. In the GR and AR assays, five and all of the 25 PCB congeners showed antagonistic activity, respectively. Among the anti-androgenic PCB congeners, the activities were more potent in PCB congeners possessing more than three chlorides including consecutive ortho- and meta- or meta- and para-chlorides. In the AhR assay using a sensitive DR-EcoScreen cell line, five of the 25 PCB congeners showed agonistic activity. We newly found that PCB-1, -35 and -56 can act as AhR agonists. Despite these activities among the PCBs, the effects of PCB-11, mainly detected in pigments and chemical products, against these receptors were found to be weaker than those of other tested PCBs. These results suggest that unintentional PCBs in pigments and chemical products might act as agonists and/or antagonists against ERα/β, AR, GR, and AhR, and some of the PCBs might disrupt endocrine functions via multiple receptors and/or simultaneously induce dioxin-like activity via AhR.

Chlorothalonil is a broad spectrum fungicide with high annual production and environmental contamination. Despite its high consumption, studies regarding the potential ecological risks of chlorothalonil, especially its metabolites, to aquatic organisms are still limited. In this study, we selected the zebrafish (Danio rerio) as the in vivo model and first identified the metabolite (4-hydroxychlorothalonil) of chlorothalonil in zebrafish by tandem quadrupole/orthogonal-acceleration time-of-flight (Q-TOF). Then, the in vivo and in vitro models were applied to comprehensively estimate the embryo toxicity and potential endocrine effect of chlorothalonil and 4-hydroxychlorothalonil. The data from zebrafish embryo toxicity showed that the lowest observed effect concentrations of both chlorothalonil and 4-hydroxychlorothalonil were 50 μg/L after 96 h of exposure. The mortality rate of the 4-hydroxychlorothalonil was 2.6-fold higher than that of the parent compound at the concentration of 50 μg/L. Dual-luciferase reporter gene assays indicated that both chlorothalonil and 4-hydroxychlorothalonil exerted estrogen receptor α (ERα) agonist activity with REC20 values of 2.4 × 10−8 M and 3.6 × 10−8 M, respectively. However, only 4-hydroxychlorothalonil exhibited both thyroid receptor β (TRβ) agonistic and antagonistic activities. Lastly, we employed molecular docking to predict the binding affinity of chlorothalonil and 4-hydroxychlorothalonil with ERα or TRβ. The results revealed that the potential endocrine effect of chlorothalonil and 4-hydroxychlorothaloni might be attributed to the different binding affinities with the receptors. In conclusion, our studies revealed that 4-hydroxychlorothalonil exhibited potent endocrine-disrupting effects compared to its parent compound, chlorothalonil. The results provided here remind us that the assessment of the potential ecological and health risks of the metabolites of fungicides in addition to their parent compounds should arouse great concerns.

Effect-directed analysis (EDA) was performed to identify the major aryl hydrocarbon receptor (AhR) agonists in sediments collected from a highly industrialized area (Lake Shihwa, Korea). Great AhR-mediated potencies were found in fractions containing aromatic compounds with log Kow values of 5–8, and relatively great concentrations of styrene oligomers (SOs) and polycyclic aromatic hydrocarbons (PAHs) were detected in those fractions. Until now, there was little information on occurrences and toxic relative potencies (RePs) of SOs in coastal environments. In the present study; i) distributions and compositions, ii) AhR binding affinities, and iii) contributions of SOs to total AhR-mediated potencies were determined in coastal sediments. Elevated concentrations of 10 SOs were detected in sediments of inland creeks ranging from 61 to 740 ng g−1 dry mass (dm), while lesser concentrations were found in inner (mean = 33 ng g−1 dm) and outer regions (mean = 25 ng g−1 dm) of the lake. Concentrations of PAHs in sediments were comparable to those of SOs. 2,4-diphenyl-1-butene (SD3) was the predominant SO analogue in sediments. SOs and PAHs were accumulated in sediments near sources, and could not be transported to remote regions due to their hydrophobicity. RePs of 3 SOs could be derived, which were 1000- to 10,000-fold less than that of one representative potent AhR active PAH, benzo[a]pyrene. Although concentrations of SOs in sediments were comparable to those of PAHs, the collective contribution of SOs to total AhR-mediated potencies were rather small (<1%), primarily due to their smaller RePs. Overall, the present study provides information on distributions and AhR binding affinities for SOs as baseline data for degradation products of polystyrene plastic in the coastal environment.