Ethanolic extract of Pyrrosia lingua (Thunb.) Farw. ameliorates OVX-induced bone loss and RANKL-induced osteoclastogenesis.

Pyrrosia lingua (Thunb.) Farw is a common plant that has been widely used as a traditional herbal medicine in China and Korea to treat patients suffering from pain, vaginal bleeding and urolithiasis. However, the pharmacological effects of P. lingua on bone remain unknown. We investigated the anti-osteoporotic effects of an ethanolic extract of P. lingua (EEPL). We found that EEPL suppressed osteoclast differentiation by directly acting on osteoclast precursor cells. EEPL suppressed the expression of receptor activator of nuclear factor-κB ligand (RANKL)-induced nuclear factor of activated T cells 1, a major transcription factor for osteoclastogenesis, by inhibiting RANKL-induced expression of aryl hydrocarbon receptor/c-Fos, and activation of nuclear factor-κB and mitogen-activated protein kinases. Moreover, administration of EEPL inhibited trabecular bone loss and weight gain in ovariectomized mice. Furthermore, we identified phytochemicals in EEPL that are known to exert anti-osteoclastogenic or anti-osteoporotic effects using ultra-high-performance liquid chromatography-tandem mass-spectrometry analysis. Overall, the results of this study suggest that EEPL is effective therapeutic candidate that can be used to prevent or treat postmenopausal osteoporosis.

Effects of 1,25-dihydroxyvitamin D3 on experimental periodontitis and AhR/NF-κB/NLRP3 inflammasome pathway in a mouse model.

Vitamin D has been known to have important regulatory functions in inflammation and immune response and shows inhibitory effects on experimental periodontitis in animal models. However, the potential mechanism has yet to be clarified. Recent studies have highlighted Aryl hydrocarbon receptor (AhR) and its downstream signaling as a crucial regulator of immune homeostasis and inflammatory regulation. OBJECTIVE: This study aimed to clarify the effect of 1,25-dihydroxyvitamin D3 (VD3) on experimental periodontitis and AhR/nuclear factor-κB (NF-κB)/NLR pyrin domain-containing 3 (NLRP3) inflammasome pathway in the gingival epithelium in a murine model. METHODOLOGY: We induced periodontitis in male C57BL/6 wild-type mice by oral inoculation of Porphyromonas gingivalis (P. gingivalis), and subsequently gave intraperitoneal VD3 injection to the mice every other day for 8 weeks. Afterwards, we examined the alveolar bone using scanning electron microscopy (SEM) and detected the gingival epithelial protein using western blot analysis and immunohistochemical staining. RESULTS: SEM images demonstrated that alveolar bone loss was reduced in the periodontitis mouse model after VD3 supplementation. Western blot analyses and immunohistochemical staining of the gingival epithelium showed that the expression of vitamin D receptor, AhR and its downstream cytochrome P450 1A1 were enhanced upon VD3 application. Additionally, VD3 decreased NF-κB p65 phosphorylation, and NLRP3, apoptosis-associated speck-like protein, caspase-1, interleukin-1ß (IL-1ß) and IL-6 protein expression. CONCLUSIONS: These results implicate the alleviation of periodontitis and the alteration of AhR/NF-κB/NLRP3 inflammasome pathway by VD3 in the mouse model. The attenuation of this periodontal disease may correlate with the regulation of AhR/NF-κB/NLRP3 inflammasome pathway by VD3.

Water-Soluble Extract from Actinidia arguta (Siebold & Zucc.) Planch. ex Miq. and Perilla frutescens (L.) Britton, ACTPER, Ameliorates a Dry Skin-Induced Itch in a Mice Model and Promotes Filaggrin Expression by Activating the AhR Signaling in HaCaT Cells.

With a complex etiology involving multiple factors, the condition known as itch is a primary symptom of many skin diseases. Current treatment methods are ineffective for addressing itches caused by dry skin, for example. We developed a botanical extract, ACTPER, made from a mixture of Actinidia arguta and Perilla frutescens, which have traditionally been used to treat itch. The quality of ACTPER as a research agent was controlled in our experiment by cell-based bioassays, as well as by high-performance liquid chromatography (HPLC), using two chemical markers. In the acetone-induced dry skin mice model, the oral administration of ACTPER alleviated dry skin-related skin properties and itching behavior. The RNA and protein expression of the filament aggregating protein (filaggrin) gene, a key factor involved in the regulation of skin barrier function, was significantly increased, as measured by quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence assay. To understand the underlying mechanism(s) at the molecular level, HaCaT cells, a human keratinocyte-derived cell line, were treated with various concentrations of ACTPER. We found that the protein expression of filaggrin was indeed upregulated by ACTPER in a dose dependent manner. Data from experiments involving the reporter plasmid containing the xenobiotic response element (XRE), and the chemical antagonist for the aryl hydrocarbon receptor (AhR), indicated that the ACTPER-mediated upregulation of filaggrin was controlled through the activation of the AhR signaling pathway. The molecular docking simulation study predicted that ACTPER might contain chemical compounds that bind directly to AhR. Taken together, our results suggest that ACTPER may provide the platform, based upon which a variety of safe and effective therapeutic agents can be developed to treat itch.

Effects of 1, 25-dihydroxyvitamin D3 on experimental periodontitis and AhR/NF-κB/NLRP3 inflammasome pathway in a mouse model

Abstract Vitamin D has been known to have important regulatory functions in inflammation and immune response and shows inhibitory effects on experimental periodontitis in animal models. However, the potential mechanism has yet to be clarified. Recent studies have highlighted Aryl hydrocarbon receptor (AhR) and its downstream signaling as a crucial regulator of immune homeostasis and inflammatory regulation. Objective: This study aimed to clarify the effect of 1,25-dihydroxyvitamin D3 (VD3) on experimental periodontitis and AhR/nuclear factor-κB (NF-κB)/NLR pyrin domain-containing 3 (NLRP3) inflammasome pathway in the gingival epithelium in a murine model. Methodology: We induced periodontitis in male C57BL/6 wild-type mice by oral inoculation of Porphyromonas gingivalis (P. gingivalis), and subsequently gave intraperitoneal VD3 injection to the mice every other day for 8 weeks. Afterwards, we examined the alveolar bone using scanning electron microscopy (SEM) and detected the gingival epithelial protein using western blot analysis and immunohistochemical staining. Results: SEM images demonstrated that alveolar bone loss was reduced in the periodontitis mouse model after VD3 supplementation. Western blot analyses and immunohistochemical staining of the gingival epithelium showed that the expression of vitamin D receptor, AhR and its downstream cytochrome P450 1A1 were enhanced upon VD3 application. Additionally, VD3 decreased NF-κB p65 phosphorylation, and NLRP3, apoptosis-associated speck-like protein, caspase-1, interleukin-1β (IL-1β) and IL-6 protein expression. Conclusions: These results implicate the alleviation of periodontitis and the alteration of AhR/NF-κB/NLRP3 inflammasome pathway by VD3 in the mouse model. The attenuation of this periodontal disease may correlate with the regulation of AhR/NF-κB/NLRP3 inflammasome pathway by VD3.

Indigo Naturalis ameliorates murine dextran sodium sulfate-induced colitis via aryl hydrocarbon receptor activation.

BACKGROUND: Indigo Naturalis (IN) is used as a traditional herbal medicine for ulcerative colitis (UC). However, the mechanisms of action of IN have not been clarified. We aimed to evaluate the efficacy of IN for ameliorating colonic inflammation. We further investigated the mechanisms of action of IN. METHODS: Colitis severity was assessed in dextran sodium sulfate-induced colitis and trinitrobenzene sulfonic acid-induced colitis models with or without the oral administration of IN or indigo, which is a known major component of IN. Colonic lamina propria (LP) mononuclear cells isolated from IN-treated mice were analyzed with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and flow cytometry. LP and splenic mononuclear cells cultured in vitro with IN or indigo were also analyzed. The role of the candidate receptor for indigo, the aryl hydrocarbon receptor (AhR), was analyzed using Ahr-deficient mice. RESULTS: Colitis severity was significantly ameliorated in the IN and indigo treatment groups compared with the control group. The mRNA expression levels of interleukin (Il)-10 and Il-22 in the LP lymphocytes were increased by IN treatment. The treatment of splenocytes with IN or indigo increased the expression of anti-inflammatory cytokines and resulted in the expansion of IL-10-producing CD4+ T cells and IL-22-producing CD3-RORγt+ cells, but not CD4+Foxp3+ regulatory T cells. The amelioration of colitis by IN or indigo was abrogated in Ahr-deficient mice, in association with diminished regulatory cytokine production. CONCLUSIONS: IN and indigo ameliorated murine colitis through AhR signaling activation, suggesting that AhR could be a promising therapeutic target for UC.

Antioxidant Opuntia ficus-indica Extract Activates AHR-NRF2 Signaling and Upregulates Filaggrin and Loricrin Expression in Human Keratinocytes.

Opuntia ficus-indica (OFI) is a cactus species widely used as an anti-inflammatory, antilipidemic, and hypoglycemic agent. It has been shown that OFI extract (OFIE) inhibits oxidative stress in animal models of diabetes and hepatic disease; however, its antioxidant mechanism remains largely unknown. In this study, we demonstrated that OFIE exhibited potent antioxidant activity through the activation of nuclear factor erythroid 2-related factor 2 (NRF2) and the downstream antioxidant enzyme NAD(P)H: quinone oxidoreductase 1 (NQO1), which inhibited the generation of reactive oxygen species in keratinocytes challenged with tumor necrosis factor α or benzo[α]pyrene. The antioxidant capacity of OFIE was canceled in NRF2 knockdown keratinocytes. OFIE exerted this NRF2-NQO1 upregulation through activation of the aryl hydrocarbon receptor (AHR). Moreover, the ligation of AHR by OFIE upregulated the expression of epidermal barrier proteins: filaggrin and loricrin. OFIE also prevented TH2 cytokine-mediated downregulation of filaggrin and loricrin expression in an AHR-dependent manner because it was canceled in AHR knockdown keratinocytes. Antioxidant OFIE is a potent activator of AHR-NRF2-NQO1 signaling and may be beneficial in treating barrier-disrupted skin disorders.

Cynaropicrin attenuates UVB-induced oxidative stress via the AhR-Nrf2-Nqo1 pathway.

Due to its antioxidant and anti-inflammatory activities, artichoke (Cynara scolymus) has been used as folk medicine to treat various diseases. Cynaropicrin (Cyn), a sesquiterpene lactone, is the major bioactive phytochemical in the artichoke; however, its pharmacological mechanism remains unknown. Because some phytochemicals exert their antioxidant activity by activating aryl hydrocarbon receptor (AhR), leading to subsequent induction of the antioxidant pathway including nuclear factor E2-related factor 2 (Nrf2) and NAD(P)H: quinone oxidoreductase 1 (Nqo1), we investigated whether Cyn also activates the AhR-Nrf2-Nqo1 pathway. Cyn indeed induced the activation (nuclear translocation) of AhR, leading to nuclear translocation of Nrf2 and dose-dependent upregulation of Nrf2 and Nqo1 mRNAs in human keratinocytes. The Cyn-induced AhR-Nrf2-Nqo1 activation was AhR- and Nrf2-dependent, as demonstrated by the observation that it was absent in keratinocytes transfected by siRNA against either AhR or Nrf2. In accordance with these findings, Cyn actively inhibited generation of reactive oxygen species from keratinocytes irradiated with ultraviolet B (UVB) in a Nrf2-dependent manner. Cyn also inhibited the production of proinflammatory cytokines such as interleukin 6 and tumor necrosis factor-α from UVB-treated keratinocytes. Our findings demonstrate that Cyn is a potent activator of the AhR-Nrf2-Nqo1 pathway, and could therefore be applied to prevention of UVB-induced photo aging.

Resveratrol and endometrium: a closer look at an active ingredient of red wine using in vivo and in vitro models.

Resveratrol is a natural phytoestrogen with antiproliferative properties present in red wine, grapes, and berries. Published reports on the effects of resveratrol in human endometrial function are limited. The objective of this study was to investigate the expression of estrogen receptor α (ESR1), Ki-67 (a proliferative marker), aryl hydrocarbon receptor (AhR), and members of the cytochrome P450 superfamily of enzymes (CYP1A1 and CYP1B1) in an in vitro and vivo assay. Alkaline phosphatase assay of estrogenicity was used to compare estrogen activity of different concentrations of resveratrol to estradiol (E2) and diethylstilbestrol (DES), using Ishikawa cell culture. Immunohistochemical expression of ESR1 and Ki67, and reverse transcriptase polymerase chain reaction of AhR, CYP1A1, and CYP1B1 were analyzed from xenograft implants of human endometrial tissue in ovariectomized immunodeficient RAG-2-γ(c) mice, after 30 days of treatment with subcutaneous pellets of E2, E2 plus progesterone (P4), or E2 plus resveratrol (6, 30, or 60 mg) for 30 days. Compared to E2, resveratrol acted as an agonist and antagonist of estrogen in low and high concentrations, respectively, when combined with E2. Xenografts of human endometrial tissues in RAG-2 mice exhibited reduced expression of ESR1 and proliferative activity (Ki67) with 60 mg of resveratrol. This study suggests that resveratrol, at high doses, has the potential benefit to reduce proliferation of human endometrium through ESR1.

Effects of anthocyanins on the AhR-CYP1A1 signaling pathway in human hepatocytes and human cancer cell lines.

Anthocyanins are plant pigments occurring in flowers and berry fruits. Since a phenomenon of food-drug interactions is increasingly emerging, we examined the effects of 21 major anthocyanins and the extracts from 3 food supplements containing anthocyanins on the aryl hydrocarbon receptor (AhR)-cytochrome P450 CYP1A1 signaling pathway in human hepatocytes and human hepatic HepG2 and intestinal LS174T cancer cells. Pelargonidin-3-O-rutinoside (PEL-2) and cyanidin-3,5-O-diglucoside (CYA-3) dose-dependently activated AhR, as revealed by gene reporter assay. PEL-2 and CYA-3 induced CYP1A1 mRNA but not protein in HepG2 and LS174T cells. Neither compounds induced CYP1A1 mRNA and protein in four different primary human hepatocytes cultures. The effects of PEL-2 and CYA-3 on AhR occurred by ligand-dependent and ligand-independent mechanisms, respectively, as demonstrated by ligand binding assay. In a direct enzyme inhibition assay, none of the antocyanins tested inhibited the CYP1A1 marker activity to less than 50% even at 100 µM concentration. PEL-2 and CYA-3 at 100 µM inhibited CYP1A1 to 79% and 65%, respectively. In conclusion, with exception of PEL-2 and CYA-3, there were no effects of 19 major anthocyanins and 3 food supplements containing anthocyanins on AhR-CYP1A1 signaling, implying zero potential of these compounds for food-drug interactions with respect to AhR-CYP1A1 pathway.

Old King coal - molecular mechanisms underlying an ancient treatment for atopic eczema.

Traditional remedies for common disorders have been known for centuries, but insight into their mechanism of action is often limited. In this issue of the JCI, Joost Schalkwijk's research group at the Radboud University Nijmegen Medical Centre in The Netherlands advances our understanding of why topical coal tar is an effective treatment for atopic dermatitis (AD), both rationalizing the use of this traditional medicine, and providing the scientific basis for new therapeutic approaches.

Ahrd Cyp1a2(-/-) mice show increased susceptibility to PCB-induced developmental neurotoxicity.

Polychlorinated biphenyls (PCBs) are developmental neurotoxicants that produce cognitive and behavioral changes in children exposed during gestation and lactation. Coplanar PCBs bind the aryl hydrocarbon receptor (AHR) and can be sequestered in liver by cytochrome P450 1A2 (CYP1A2). The AHR is a ligand-activated transcription factor which increases expression of the CYP1 family, including CYP1A2. Our previous work examining genetic susceptibility to developmental PCB neurotoxicity showed that Ahr(b)Cyp1a2(-/-) mice with the high-affinity Ahr(b) allele and lacking CYP1A2 were most susceptible while Ahr(b)Cyp1a2(+/+) and poor-affinity Ahr(d)Cyp1a2(+/+) mice were resistant. To follow up, a fourth line of mice was generated with the Ahr(d)Cyp1a2(-/-) genotype and compared with the background strain Ahr(b)Cyp1a2(+/+). Dams received a PCB mixture or the corn oil vehicle at gestational Day 10 (GD10) and postnatal Day 5 (PND5). Offspring were tested at PND60 in open field locomotor, acoustic startle with pre-pulse inhibition (PPI), novel object recognition and Morris water maze. Locomotor activity was increased in PCB-treated Ahr(b)Cyp1a2(+/+) mice, but no differences were seen in control vs. PCB-treated Ahr(d)Cyp1a2(-/-) mice. PCB-treated Ahr(d)Cyp1a2(-/-) mice had a higher baseline startle response and significantly reduced pre-pulse inhibition at the 74dB level compared with corn oil-treated controls (P<0.05). PCB-treated Ahr(d)Cyp1a2(-/-) mice had impairments in novel objective recognition (P<0.05) and during all three hidden platform phases of Morris water maze (P<0.01). Combined with our previous findings, these results indicate Cyp1a2 genotype is more important in susceptibility to PCB-induced deficits in learning and memory, but Ahr genotype appears more important when assessing acoustic startle-PPI and locomotor activity.

Modified expression of aryl hydrocarbon receptor-related genes by deoxymiroestrol, a phytoestrogen, in mouse hepatocytes in primary culture.

ETHNOPHARMACOLOGICAL RELEVANCE: Deoxymiroestrol (DM), a strong phytoestrogen from Pueraria candollei Wall. ex Benth. var. mirifica (family Leguminosae). This plant has long been used in traditional medicine for rejuvenation. MATERIALS AND METHODS: The expression of aryl hydrocarbon receptor-related genes in mouse hepatocytes in primary culture was quantified by real-time RT-PCR and hepatic microsomal P450 activity was assessed by using ethoxyresorufin O-dealkylation. RESULTS: The mRNA expression of the aryl hydrocarbon receptor (AhR), AhR nuclear translocator, and CYP1A1 was suppressed, whereas that of CYP1B1, estrogen receptor α (ERα), CYP2B9, and glutathione-S-transferase a2 (GSTa2) was increased. The effects of DM on the gene expression depended on treatment period and concentrations, and were similar to those of ß-estradiol (E2). DM and E2 at pharmacological concentrations had a marked synergistic effect on CYP1A1 expression after combined treatment with a typical CYP1 inducer, ß-naphthoflavone (ßNF), at the level of both transcription and enzymatic activity. DM enhanced the inducible mRNA expression of CYP1A1 and CYP1B1 similar to E2. Meanwhile, the expression of ERα mRNA was not affected by ßNF, which, on the contrary, completely eliminated the DM-induced mRNA expression of ERα, CYP2B9, and GSTa2. CONCLUSION: The findings that DM modified the expression of several metabolism-related genes suggest the need for caution when using health supplements having phytoestrogenic activity.

Effect of beta-naphthoflavone on AhR-regulated genes (CYP1A1, 1A2, 1B1, 2S1, Nrf2, and GST) and antioxidant enzymes in various brain regions of pig.

The constitutive and inducible expression of aryl hydrocarbon receptor (AhR) and of the AhR-regulated genes coding for CYP1A1, CYP1A2, CYP1B1, CYP2S1, and Nrf2 was investigated by real-time or traditional PCR in cerebral areas (cortex, cerebellum, midbrain, and hippocampus), blood-brain interfaces (meninges and brain microvessels) and liver obtained from control pigs and from pigs treated with beta-naphthoflavone (betaNF), a potent AhR agonist. The enzymatic activities of ethoxyresorufin-O-deethylase (EROD), and methoxyresorufin-O-deethylase (MEROD), marker for CYP1A1 and CYP1A2, the GST and various antioxidant enzymes (catalase, superoxide dismutase, GSSG-reductase, and GSH-peroxidase) were also determined in the same CNS regions. The AhR, CYP1A1, CYP1A2, CYP1B1, Nrf2 mRNAs were detected, although at different extent, in all the CNS regions, while CYP2S1 mRNA was detected only in midbrain. In the blood-brain interfaces, the constitutive basal expression of AhR and CYP1A1 was comparable to the hepatic one and even higher for CYP1B1 and Nrf2. The treatment with betaNF determined the induction of CYP1A1 and 1B1 (but not of AhR, CYP1A2, and Nrf2) mRNA levels in various CNS areas; notably, CYP1A1 mRNA was increased to about 300-fold in the microvessels. The analysis of enzymatic activities revealed that EROD, but not MEROD, was induced in microsomes but not in mitochondria of all the CNS areas. However, the mitochondrial EROD activities were comparable (in midbrain, meninges) or higher (in cortex, cerebellum, hippocampus) than the microsomal ones, suggesting an important metabolic function of CYP1A1 in this subcellular localization. The activities of GST and antioxidant enzymes were detected in all CNS tissues, with levels lower than the hepatic ones, but found quite evenly distributed and marginally affected by betaNF treatment. The high expression of metabolic enzymes found in blood-brain interfaces could represent a very important defence toward toxins of CNS.

Comparison of hormonal activity of isoflavone-containing supplements used to treat menopausal complaints.

OBJECTIVES: The isoflavones present in red clover and soy are used as an alternative treatment for menopausal complaints and are commercially available as high-dose food supplements. These preparations contain varying amounts of active ingredients, often without detailed specifications. Thus, it is difficult to derive a recommended daily dose, and the reliability of these products is rather low. METHODS: We quantified the isoflavone content of 19 different isoflavone-containing preparations and compared their binding and transactivational activities with regard to estrogen receptor alpha, estrogen receptor beta, androgen receptor, progesterone receptor, peroxisome-proliferator-activated receptor, and aryl hydrocarbon receptor. RESULTS: The food supplements that we tested bound to and transactivated both the estrogen receptors and the other receptors. After comparing the isoflavone content quantified by us with the isoflavone content specified on the package labels, we found that at least the specified isoflavone content or more could be detected in only 5 of the 19 food supplements that we tested. CONCLUSIONS: Preparations containing isoflavones should be standardized for the isoflavone aglycone content to facilitate the prediction of theoretical hormonal activity, facilitate the intake of a controlled amount of isoflavones, and ensure greater product reliability.

Bioactive terpenoids and flavonoids from Ginkgo biloba extract induce the expression of hepatic drug-metabolizing enzymes through pregnane X receptor, constitutive androstane receptor, and aryl hydrocarbon receptor-mediated pathways.

PURPOSE: The objective of the current study is to investigate the hypothesis that bioactive terpenoids and flavonoids of Ginkgo biloba extract (GBE) induce human hepatic drug metabolizing enzymes (DMEs) and transporters through the selective activation of pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR). METHODS: Human primary hepatocyte (HPH), and HepG2 cells are used as in vitro models for enzyme induction and nuclear receptor activation studies. A combination of real-time RT-PCR, transient transfection, and cell-based reporter assays were employed. RESULTS: In human primary hepatocytes, real-time PCR analysis showed induction of CYP2B6, CYP3A4, UGT1A1, MDR1, and MRP2 by EGb 761, ginkgolide A (GA) and ginkgolide B (GB), but not by bilobalide (BB) or the flavonoids (quercetin, kaempferol and tamarixetin) of GBE. Cell-based reporter assays in HepG2 revealed that GA and GB are potent activators of PXR; quercetin and kaempferol activate PXR, CAR, and AhR, whereas BB exerts no effects on these xenobiotic receptors. Notably, the flavonoids induced the expression of UGT1A1 and CYP1A2 in HepG2 cells but not in HPH. CONCLUSION: Our results indicate that terpenoids and flavonoids of GBE exhibit differential induction of DMEs through the selective activation of PXR, CAR, and AhR.

Induction of cytochrome P450 1A1 expression by ginsenoside Rg1 and Rb1 in HepG2 cells.

Transcriptional activation of the human CYP1A1 gene (coding for cytochrome P450 1A1) is mediated by the aryl hydrocarbon receptor. In the present study we have examined interaction of the ginsenoside Rg1 and Rb1 with the carcinogen activation pathway mediated by the aryl hydrocarbon receptor in HepG2 cells. RT-PCR was used to determine the CYP1A1 mRNA levels. The results showed that in HepG2 cells CYP1A1 mRNA expression was significantly increased in a concentration- and time- dependent manner by ginsenoside Rg1 and Rb1. Ginsenoside Rg1 and Rb1 activated the DNA-binding capacity of the aryl hydrocarbon receptor for the xenobiotic responsive element of CYP1A1 as measured by the electrophoretic-mobility shift assay (EMSA). Rg1 and Rb1 were able to activate the ability of the aryl hydrocarbon receptor to bind to an oligonucleotide containing the xenobiotic-responsive element (XRE) of the cyp1a1 promoter. These results indicate that Rg1 and Rb1's effects on CYP1A1 induction are mediated by the aryl hydrocarbon receptor. Since CYP1A1 and aryl hydrocarbon receptor play important roles in carcinogenesis, development, differentiation and many other essential physiological functions, these results suggest that the chemopreventive effect of Panax ginseng may be due, in part, to ginsenoside Rg1 and Rb1's ability to compete with aryl hydrocarbons for both the aryl hydrocarbon receptor and CYP1A1. Rg1 and Rb1 may thus be natural ligands and substrates of the aryl hydrocarbon receptor or have relationship with aryl hydrocarbon receptor pathway. These properties might be of help for future studies in P. ginseng and chemoprevention in chemical-induced cancer.

Assessing the potential risk of oil-field produced waters using a battery of bioassays/biomarkers.

A battery of in vitro bioassays was conducted to assess the potential risks of organic extracts from oilfield produced wastewaters and from the receiving waters. In SOS/umu bioassay for the genotoxicity, our results showed that direct and indirect genotoxic substances were observed and metabolic activation greatly enhanced the genotoxic effects. In Ethoxyresorufin-O-deethylase bioassay, levels of AhR-agonists, expressed as 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalences, varied from 13.3 to 16.7 pg L(-1). We conclude that oilfield produced wastewater contains substantial quantity of indirect genotoxic substances exclusive of AhR agonists. Both genotoxic and AhR agonistic chemicals could not be effectively removed by the treatment processes.

Low-dose dioxins alter gene expression related to cholesterol biosynthesis, lipogenesis, and glucose metabolism through the aryl hydrocarbon receptor-mediated pathway in mouse liver.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a common environmental contaminant. TCDD binds and activates the transcription factor aryl hydrocarbon receptor (AHR), leading to adverse biological responses via the alteration of the expression of various AHR target genes. Although small amounts of TCDD are consumed via contaminated daily foodstuffs and environmental exposures, the effects of low-dose TCDD on gene expression in animal tissues have not been clarified, while a number of genes affected by high-dose TCDD were reported. In this study, we comprehensively analyzed gene expression profiles in livers of C57BL/6N mice that were orally administered relatively low doses of TCDD (5, 50, or 500 ng/kg body weight (bw) day(-1)) for 18 days. The hepatic TCDD concentrations, measured by gas chromatography-mass spectrometry, were 1.2, 17, and 1063 pg toxicity equivalent quantity (TEQ)/g, respectively. The mRNA level of the cytochrome P450 CYP1A1 was significantly increased by treatment with only TCDD 500 ng/kg bw day(-1). DNA microarray and quantitative RT-PCR analyses revealed changes in the expression of genes involved in the circadian rhythm, cholesterol biosynthesis, fatty acid synthesis, and glucose metabolism in the liver with at all doses of TCDD employed. However, repression of expression of genes involved in energy metabolism was not observed in the livers of Ahr-null mice that were administered the same dose of TCDD. These results indicate that changes in gene expression by TCDD are mediated by AHR and that exposure to low-dose TCDD could affect energy metabolism via alterations of gene expression.

Mechanisms of interaction of endocrine-disrupting chemicals with glutamate-evoked secretion of gonadotropin-releasing hormone.

In previous studies, we detected a dichlorodiphenyltrichloroethane (DDT) derivative in the serum of children with sexual precocity after migration from developing countries. Recently, we reported that DDT stimulated pulsatile gonadotropin-releasing hormone (GnRH) secretion and sexual maturation in the female rat. The aim of this study was to delineate the mechanisms of interaction of endocrine-disrupting chemicals including DDT with GnRH secretion evoked by glutamate in vitro. Using hypothalamic explants obtained from 15-day-old female rats, estradiol (E2) and DDT caused a concentration-related increase in glutamate-evoked GnRH release while p,p'-dichlorodiphenyldichloroethene and methoxychlor had no effect. The effective DDT concentrations in vitro were consistent with the serum concentrations measured in vivo 5 days after exposure of immature rats to 10 mg/kg/day of o,p'-DDT. Bisphenol A induced some stimulatory effect, whereas no change was observed with 4-nonylphenol. The o,p'-DDT effects in vitro were prevented partially by a selective antagonist of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of glutamate receptors. A complete prevention of o,p'-DDT effects was caused by an estrogen receptor (ER) antagonist as well as an aryl hydrocarbon receptor (AHR) antagonist and inhibitors of protein kinases A and C and mitogen-activated kinases. While an intermittent incubation with E2 caused no change in amplification of the glutamate-evoked GnRH release for 4 h, continuous incubation with E2 or o,p'-DDT caused an increase of this amplification after 3.5 h of incubation. In summary, DDT amplifies the glutamate-evoked GnRH secretion in vitro through rapid and slow effects involving ER, AHR, and AMPA receptor mediation.

Influence of food polyphenols on aryl hydrocarbon receptor-signaling pathway estimated by in vitro bioassay.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxic and biological actions of many aromatic environmental pollutants such as dioxins. We investigated AhR activation by some vegetable constituents, including flavonoids, tannins, and related polyphenols, using an AhR-based in vitro bioassay for dioxins. Among the compounds tested, marked AhR activation was exhibited by isoflavones such as daidzein, resveratrol (a stilbene) structure, some flavanones such as naringenin, and flavones such as baicalein. On the other hand, some flavones such as apigenin, flavonols such as quercetin, and anthraquinones such as emodin, showed notable inhibitory effects on the in vitro activation of AhR induced by the dioxin [2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)]. In addition, AhR-mediated interactions between AhR and some plant extracts, including those from vegetables, fruits, herbs, and teas, were tested by using the AhR-based bioassay. Of the samples tested, some leafy green vegetables, citrus fruits, and herbs that contain food polyphenolics showed AhR-based interactions at high concentrations. On the basis of these finding, we discuss the implications of polyphenols on the AhR-signaling pathway.