Aryl Hydrocarbon Receptor Signal Transduction Research Articles

Characterization of the Fundulus heteroclitus embryo transcriptional response and development of a gene expression-based fingerprint of exposure for the alternative flame retardant, TBPH (bis (2-ethylhexyl)-tetrabromophthalate)

Although alternative Flame Retardant (FR) chemicals are expected to be safer than the legacy FRs they replace, their risks to human health and the environment are often poorly characterized. This study used a small volume, fish embryo system to reveal potential mechanisms of action and diagnostic exposure patterns for TBPH (bis (2-ethylhexyl)-tetrabromophthalate), a component of several widely-used commercial products. Two different concentration of TBPH were applied to sensitive early life stages of an ecologically important test species, Fundulus heteroclitus (Atlantic killifish), with a well-annotated genome. Exposed fish embryos were sampled for transcriptomics or chemical analysis of parent compound and primary metabolite or observed for development and survival through larval stage. Global transcript profiling using RNA-seq was conducted (n = 16 per treatment) to provide a non-targeted and statistically robust approach to characterize TBPH gene expression patterns. Transcriptomic analysis revealed a dose-response in the expression of genes associated with a surprisingly limited number of biological pathways, but included the aryl hydrocarbon receptor signal transduction pathway, which is known to respond to several toxicologically-important chemical classes. A transcriptional fingerprint using Random Forests was developed that was able to perfectly discriminate exposed vs. non-exposed individuals in test sets. These results suggest that TBPH has a relatively low potential for developmental toxicity (at least in fishes), despite concerns related to its structural similarities to endocrine disrupting chemicals and that the early life stage Fundulus system may provide a convenient test system for exposure characterization. More broadly, this study advances the usefulness of a biological testing and analysis system utilizing non-targeted transcriptomics profiling and early developmental endpoints that complements current screening methods to characterize chemicals of ecological and human health concern.

Endosulfan Induces CYP1A1 Expression Mediated through Aryl Hydrocarbon Receptor Signal Transduction by Protein Kinase C.

CYP1A1 is a phase I xenobiotic-metabolizing enzyme whose expression is mainly driven by AhR. Endosulfan is an organochlorine pesticide used agriculturally for a wide range of crops. In this study, we investigated the effect of endosulfan on CYP1A1 expression and regulation. Endosulfan significantly increased CYP1A1 enzyme activity as well as mRNA and protein levels. In addition, endosulfan markedly induced XRE transcriptional activity. CH-223191, an AhR antagonist, blocked the endosulfan-induced increase in CYP1A1 mRNA and protein expression. Moreover, endosulfan did not induce CYP1A1 gene expression in AhR-deficient mutant cells. Furthermore, endosulfan enhanced the phosphorylation of calcium calmodulin (CaM)-dependent protein kinase (CaMK) and protein kinase C (PKC). In conclusion, endosulfan-induced up-regulation of CYP1A1 is associated with AhR activation, which may be mediated by PKC-dependent pathways.

Ginsenosides are novel naturally-occurring aryl hydrocarbon receptor ligands.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of structurally diverse chemicals. In this study, we examined the ability of a series of ginsenosides extracted from ginseng, a traditional Chinese medicine, to bind to and activate/inhibit the AHR and AHR signal transduction. Utilizing a combination of ligand and DNA binding assays, molecular docking and reporter gene analysis, we demonstrated the ability of selected ginsenosides to directly bind to and activate the guinea pig cytosolic AHR, and to stimulate/inhibit AHR-dependent luciferase gene expression in a recombinant guinea pig cell line. Comparative studies revealed significant species differences in the ability of ginsenosides to stimulate AHR-dependent gene expression in guinea pig, rat, mouse and human cell lines. Not only did selected ginsenosides preferentially activate the AHR from one species and not others, mouse cell line was also significantly less responsive to these chemicals than rat and guinea pig cell lines, but the endogenous gene CYP1A1 could still be inducted in mouse cell line. Overall, the ability of these compounds to stimulate AHR signal transduction demonstrated that these ginsenosides are a new class of naturally occurring AHR agonists.

ShRNA-mediated gene silencing of AHR promotes the differentiation of P19 mouse embryonic carcinoma cells into cardiomyocytes

The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix (bHLH) transcription factor that is activated by environmental contaminants including polychlorinated biphenyls (PCBs). The AHR affects a variety of processes that are involved in cell growth and differentiation. In this study, we constructed a P19 embryonic carcinoma cell line with AHR gene silencing using the vector-based approach of short hairpin (sh)RNA interference that allows cells to differentiate into cardiac myocytes when treated with dimethyl sulfoxide (DMSO). The expression levels of the cardiac development-specific GATA4 and Nkx2.5 genes were measured using real-time quantitative polymerase chain reaction (qPCR). Our data showed that the expression levels of the GATA4 and Nkx2.5 genes were increased in the AHR-silenced P19 cells compared with the control groups. Four critical genes (ARNT, CYP1A1, GSK3β and β-catenin) expressed in the AHR and in the Wnt signaling pathway were also measured by qPCR. We found that the expression levels of ARNT, CYP1A1 and β-catenin were suppressed, whereas GSK3β expression was elevated in the AHR-silenced P19 cells. Therefore, it is possible that the silencing of AHR promotes the differentiation of P19 cells through the AHR and Wnt signal transduction pathway.

Involvement of aryl hydrocarbon receptor signaling in the development of small cell lung cancer induced by HPV E6/E7 oncoproteins

BackgroundLung cancers consist of four major types that and for clinical-pathological reasons are often divided into two broad categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). All major histological types of lung cancer are associated with smoking, although the association is stronger for SCLC and squamous cell carcinoma than adenocarcinoma. To date, epidemiological studies have identified several environmental, genetic, hormonal and viral factors associated with lung cancer risk. It has been estimated that 15-25% of human cancers may have a viral etiology. The human papillomavirus (HPV) is a proven cause of most human cervical cancers, and might have a role in other malignancies including vulva, skin, oesophagus, head and neck cancer. HPV has also been speculated to have a role in the pathogenesis of lung cancer. To validate the hypothesis of HPV involvement in small cell lung cancer pathogenesis we performed a gene expression profile of transgenic mouse model of SCLC induced by HPV-16 E6/E7 oncoproteins.MethodsGene expression profile of SCLC has been performed using Agilent whole mouse genome (4 × 44k) representing ~ 41000 genes and mouse transcripts. Samples were obtained from two HPV16-E6/E7 transgenic mouse models and from littermate's normal lung. Data analyses were performed using GeneSpring 10 and the functional classification of deregulated genes was performed using Ingenuity Pathway Analysis (Ingenuity® Systems, http://www.ingenuity.com).ResultsAnalysis of deregulated genes induced by the expression of E6/E7 oncoproteins supports the hypothesis of a linkage between HPV infection and SCLC development. As a matter of fact, comparison of deregulated genes in our system and those in human SCLC showed that many of them are located in the Aryl Hydrocarbon Receptor Signal transduction pathway.ConclusionsIn this study, the global gene expression of transgenic mouse model of SCLC induced by HPV-16 E6/E7 oncoproteins led us to identification of several genes involved in SCLC tumor development. Furthermore, our study reveled that the Aryl Hydrocarbon Receptor Signaling is the primarily affected pathway by the E6/E7 oncoproteins expression and that this pathway is also deregulated in human SCLC. Our results provide the basis for the development of new therapeutic approaches against human SCLC.

A system-level investigation into the cellular toxic response mechanism mediated by AhR signal transduction pathway

Viewing a cellular system as a collection of interacting parts can lead to new insights into the complex cellular behavior. In this study, we have investigated aryl hydrocarbon receptor (AhR) signal transduction pathway from such a system-level perspective. AhR detects various xenobiotics, such as drugs or endocrine disruptors (e.g. dioxin), and mediates transcriptional regulation of target genes such as those in the cytochrome P450 (CYP450) family. On binding with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), however, AhR becomes abnormally activated and conveys toxic effects on cells. Despite many related studies on the TCDD-mediated toxicity, quantitative system-level understanding of how TCDD-mediated toxicity generates various toxic responses is still lacking. Here, we present a manually curated TCDD-mediated AhR signaling pathway including crosstalks with the hypoxia pathway that copes with oxygen deficiency and the p53 pathway that induces a DNA damage response. Based on the integrated pathway, we have constructed a mathematical model and validated it through quantitative experiments. Using the mathematical model, we have investigated: (i) TCDD dose-dependent effects on AhR target genes; (ii) the crosstalk effect between AhR and hypoxia signals; and (iii) p53 inhibition effect of TCDD-liganded AhR. Our results show that cellular intake of TCDD induces AhR signaling pathway to be abnormally up-regulated and thereby interrupts other signaling pathways. Interruption of hypoxia and p53 pathways, in turn, can incur various hazardous effects on cells. Taken together, our study provides a system-level understanding of how AhR signal mediates various TCDD-induced toxicities under the presence of hypoxia and/or DNA damage in cells.

Transcriptional and posttranscriptional regulation of CYP1A1 by vanadium in human hepatoma HepG2 cells

We recently demonstrated that V(5+) downregulates 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated induction of Cyp1a1 mRNA, protein, and catalytic activity levels in Hepa 1c1c7 cells through transcriptional mechanism. Therefore, it is important to investigate whether similar changes occur in humans. For this purpose, we examined the effect of V(5+) (as ammonium metavanadate, NH(4)VO(3)) on the expression of aryl hydrocarbon receptor (AhR)-regulated gene; cytochrome P450 1A1 (CYP1A1) at each step of the AhR signal transduction pathway in human hepatoma HepG2 cells. Our results show a significant reduction in TCDD-mediated induction of CYP1A1 mRNA, protein, and activity levels after V(5+) treatment in a dose-dependent manner. Investigating the effect of co-exposure to V(5+) and TCDD at transcriptional levels revealed that V(5+) significantly inhibited TCDD-mediated induction of AhR-dependent luciferase reporter gene expression. Looking at the posttranscriptional level, V(5+) did not affect CYP1A1 mRNA stability, thus eliminating the possible role of V(5+) in modifying CYP1A1 gene expression through this mechanism. On the other hand, at the posttranslational level, V(5+) was able to significantly decrease CYP1A1 protein half-life contributing to the inconsistency between catalytic activity and transcriptional level. Importantly, we showed that V(5+) did not significantly alter the heme oxygenase-1 mRNA level, thus eliminating any possibility that V(5+) might have decreased CYP1A1 activity through affecting its heme content. This study demonstrates for the first time that V(5+) downregulates the expression of CYP1A1 at the transcriptional, posttranscriptional and posttranslational mechanisms in the human hepatoma HepG2 cells.

Modulation of CYP1A1 by PKC Inhibitors and TPA Pre-Treatments in MH1C1 Rat Hepatoma Cells Exposed to 3 -Methylcholanthrene

Cytochrome P4501A1 (CYP1A1), an enzyme known to metabolize polycyclic aromatic hydrocarbons, is regulated by the aryl hydrocarbon receptor (AhR). The involvement of protein kinase C (PKC) in the regulation of AhR signal transduction pathway, has been widely studied but the role of specific PKC isoform(s) involved in this process it is not well clarified. To study which PKC isoform(s) is implicated in the regulation of CYP1A1, in the poorly tumorigenic MH1C1 rat hepatoma cells, we examined the effects of some PKC pharmacological inhibitors, Calphostin C (CAL), Staurosporine (STA) and H7, and of 12-0-tetradecanoyl phorbol 13-acetate (TPA), a PKC activator, on basal and 3- methylcholanthrene (MC)-induced CYP1A1 protein expression and mediated ethoxyresorufin O-deethylation (EROD) activity. In parallel, the activities of PKC-α, -βI, -δ and -e isoforms, the most expressed in MH1C1 cells, were monitored. After pre-treatment with CAL, STA and H7, the MC-induced CYP1A1 protein and EROD activity were rapidly reduced with temporal profile similar to the profile of the activity of α and β1 PKC isoforms. Moreover, TPA pre-treatment induced a biphasic effect on EROD activity, and a decline of PKC -βI and -α, in first instance, and -δ and -e activities later on. These findings clearly show that, in MH1C1 cells, PKC is involved in CYP1A1 regulation and that α and βI classic PKC isoforms play an active role in modulating this process.

Role of the Per/Arnt/Sim Domains in Ligand-dependent Transformation of the Aryl Hydrocarbon Receptor

The aryl hydrocarbon receptor (AhR) mediates the toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. In a process termed transformation, ligand binding converts the AhR into its high affinity DNA binding form that represents a dimer of the AhR and Arnt, a closely related nuclear protein. During transformation, protein chaperone Hsp90 is thought to be replaced by Arnt in overlapping binding sites in the basic helix loop helix and PASB domains of the AhR. Here, analysis of AhR variants containing a modified PASB domain and AhR PASA-PASB fragments of various lengths revealed (i) an inhibitory effect on transformation concomitant with Hsp90 binding in the PASB domain, (ii) an ability of the PASA-PASB fragment of the AhR to reproduce key steps in the transformation process, and (iii) a ligand-dependent conformational change in the PASA domain consistent with increased PASA exposure during AhR transformation. Based on these results, we propose a new mechanism of AhR transformation through initiation of Arnt dimerization and Hsp90 displacement in AhR PASA/B domains. This study provides insights into mechanisms of AhR transformation, dimerization of PAS domain proteins, and Hsp90 dissociation in activation of its client proteins.

Down-Regulation of the Carcinogen-Metabolizing Enzyme Cytochrome P450 1a1 by Vanadium

Vanadium (V(5+)), a heavy metal contaminant with important toxicological consequences, has received considerable attention as an anticancer agent, although the mechanisms remain unknown. As a first step to investigate these mechanisms, we examined the effect of V(5+) (as ammonium metavanadate, NH(4)VO(3)) on the expression of the aryl hydrocarbon receptor (AhR)-regulated gene: cytochrome P450 1a1 (Cyp1a1) at each step of the AhR signal transduction pathway, using Hepa 1c1c7 cells. Our results showed a significant reduction in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated induction of Cyp1a1 mRNA, protein and activity levels after V(5+) treatments in a dose-dependent manner. Investigation of the effect of coexposure to V(5+) and TCDD at transcriptional levels revealed that V(5+) significantly inhibited TCDD-mediated induction of AhR-dependent luciferase reporter gene expression. Furthermore, despite not affecting the direct activation of the cytosolic AhR by TCDD and subsequently transforming it to a DNA-binding form, V(5+) inhibited the nuclear accumulation of liganded AhR and subsequent formation of the AhR/aryl hydrocarbon nuclear translocator (Arnt)/xenobiotic responsive element (XRE) complex. Importantly, the V(5+)-mediated inhibition of AhR/Arnt/XRE complex formation coincided with a significant decrease in ecto-ATPase activity. Looking at the post-transcriptional and post-translational effects of V(5+) on existing Cyp1a1 mRNA and protein levels, we showed that V(5+) did not affect Cyp1a1 mRNA or protein stability, thus eliminating possible role of V(5+) in modifying Cyp1a1 gene expression through these mechanisms. This study provides the first evidence that V(5+) down-regulates the expression of Cyp1a1 at the transcriptional level through an ATP-dependent mechanism.

Interaction of diuron and related substituted phenylureas with the Ah receptor pathway.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of structurally diverse chemicals, including the ubiquitous environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin. Here, we have examined the ability of diuron, a widely used herbicide, and several structurally related substituted phenylureas to bind to and activate/inhibit the AhR and AhR signal transduction. Diuron induced CYP1A1 mRNA levels in mouse hepatoma (Hepa1c1c7) cells and AhR-dependent luciferase reporter gene expression in stably transfected mouse, rat, guinea pig, and human cell lines. In addition, ligand binding and gel retardation analysis demonstrated the ability of diuron to competitively bind to and stimulate AhR transformation and DNA binding in vitro and in intact cells. Several structurally related substituted phenylureas competitively bound to the guinea pig hepatic cytosolic AhR, inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced AhR-dependent luciferase reporter gene expression in a species-specific manner and stimulated AhR transformation and DNA binding, consistent with their role as partial AhR agonists. These results demonstrate not only that diuron and related substituted phenylureas are AhR ligands but also that exposure to these chemicals could induce/inhibit AhR-dependent biological effects.

Regulation of aryl hydrocarbon receptor signal transduction by protein tyrosine kinases

The involvement of protein tyrosine kinases (PTKs) in aryl hydrocarbon receptor (AhR)-mediated signalling by omeprazole and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) was investigated in hepatoma cells. Both omeprazole- and TCDD-dependent AhR signalling was attenuated by inhibition of c- src kinase, either by using pyrazolopyrimidine 4-amino-5-(4-methylphenyl)-7-( t-butyl)pyrazolo[3,4 ]pyrimidine (PP1) and 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine (PP2) inhibitors or by expression of dominant-negative c- src. These results indicate that the overall AhR function is modulated by c- src kinase activity. In contrast, a selective inhibition of omeprazole-mediated AhR signalling was revealed by tyrosine kinase inhibitors, tyrphostins AG17 and AG879. Furthermore, omeprazole-dependent AhR activation was abolished by mutation of Tyr 320 to Phe, suggesting that this residue is a putative phosphorylation site. TCDD-dependent AhR signalling was neither affected by tyrphostins nor by this mutation. Our results are consistent with activation of the AhR by omeprazole in a ligand-independent manner, via a signal transduction pathway that involves protein tyrosine kinases, and are different from the mechanism exerted by high-affinity ligands.

Interaction networks in yeast define and enumerate the signaling steps of the vertebrate aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) is a vertebrate protein that mediates the toxic and adaptive responses to dioxins and related environmental pollutants. In an effort to better understand the details of this signal transduction pathway, we employed the yeast S. cerevisiae as a model system. Through the use of arrayed yeast strains harboring ordered deletions of open reading frames, we determined that 54 out of the 4,507 yeast genes examined significantly influence AHR signal transduction. In an effort to describe the relationship between these modifying genes, we constructed a network map based upon their known protein and genetic interactions. Monte Carlo simulations demonstrated that this network represented a description of AHR signaling that was distinct from those generated by random chance. The network map was then explored with a number of computational and experimental annotations. These analyses revealed that the AHR signaling pathway is defined by at least five distinct signaling steps that are regulated by functional modules of interacting modifiers. These modules can be described as mediating receptor folding, nuclear translocation, transcriptional activation, receptor level, and a previously undescribed nuclear step related to the receptor's Per–Arnt–Sim domain.

Induction of murine NAD(P)H:quinone oxidoreductase by 2,3,7,8-tetrachlorodibenzo-p-dioxin requires the CNC (cap 'n' collar) basic leucine zipper transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2): cross-interaction between AhR (aryl hydrocarbon receptor) and Nrf2 signal transduction.

TCDD (2,3,7,8-tetrachlorodibenzo- p -dixoin) induces phase II drug-metabolizing enzyme NQO1 [NAD(P)H:quinone oxidoreductase; EC 1.6.99.2; DT-diaphorase] in a wide range of mammalian tissues and cells. Here, we analysed the molecular pathway mediating NQO1 induction by TCDD in mouse hepatoma cells. Inhibition of protein synthesis with CHX (cycloheximide) completely blocks induction of NQO1 by TCDD as well as the basal expression and induction by phenolic antioxidant tBHQ (2-t-butylbenzene-1,4-diol), implicating a labile factor in NQO1 mRNA expression. The inhibition is both time- and concentration-dependent, requires inhibition of protein synthesis, and occurs at a transcriptional level. Inhibition of NQO1 transcription by CHX correlates with a rapid reduction of the CNC bZip (cap 'n' collar basic leucine zipper) transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) through the 26 S proteasome pathway. Moreover, blocking Nrf2 degradation with proteasome inhibitor MG132 increases the amount of Nrf2 and superinduces NQO1 in the presence of TCDD or tBHQ. Finally, genetic experiments using AhR (aryl hydrocarbon receptor)-, Arnt (aryl hydrocarbon receptor nuclear translocator)- or Nrf2-deficient cells reveal that, while induction of NQO1 by TCDD depends on the presence of AhR and Arnt, the basal and inducible expression of NQO1 by either TCDD or tBHQ requires functional Nrf2. The findings demonstrate a novel role of Nrf2 in the induction of NQO1 by TCDD and provide new insights into the mechanism by which Nrf2 regulates the induction of phase II enzymes by both phenolic antioxidants and AhR ligands.

A new luciferase reporter gene assay for the detection of androgenic and antiandrogenic effects based on a human prostate specific antigen promoter and PC3/AR human prostate cancer cells.

We developed a new mammalian cell-based luciferase reporter gene assay for androgenic and antiandrogenic activities of chemicals and environmental samples. Environmental samples usually have a complex matrix that may contain the constituents acting as androgen receptor (AR) agonists, AR antagonists or aryl hydrocarbon receptor (AhR) agonists. AhR agonists are known to elicit the antiandrogenic effect through cross-talk between AR and AhR signal transduction pathways. In this study, PC3/AR human prostate carcinoma cells were transiently transfected with a prostate-specific antigen (PSA) promoter-driven luciferase expression plasmid. The cells were treated with a test compound or an environmental sample for 24 h at 37 degrees C and then measured for luciferase activity. The luciferase activity was induced by dihydrotestosterone (DHT) in a concentration-dependent manner in a concentration range from 10 fM to 1 nM. R1881, a synthetic androgen receptor agonist, induced luciferase activity and its inductive effects was additive to that of DHT. The luciferase activity was not induced by cortisol, a glucocorticoid, progesterone, a progestin, and 17beta-estradiol, an estrogen in a concentration range of up to 1 microM. DHT-induced luciferase activity was reduced by bicalutamide and cyproterone acetate, AR antagonists, and also by benzo[a]pyrene, an aryl hydrocarbon receptor agonist, through AhR-mediated pathways. All of these findings indicate that the present assay system correctly responds to AR agonists, AR antagonists and AhR agonist and, therefore, it is a powerful tool for the sensitive and selective screening of chemicals and environmental samples for their androgenic and antiandrogenic activities. We developed the first assay system, in which the expression of luciferase was driven by the promoter of a prostate-specific antigen gene, a typical human androgen-regulated gene.

Expression and inducibility of aryl hydrocarbon receptor pathway genes in wild-caught killifish (Fundulus heteroclitus) with different contaminant-exposure histories.

Wildcaught killifish from a contaminated site on the Elizabeth River (VA, USA) are refractory to induction of cytochrome P4501A (CYP1A, measured as catalytic activity and immunodetectable CYP1A protein) after exposure to typical aryl hydrocarbon receptor (AHR) agonists, as has been reported for fish from other sites highly contaminated with these compounds. In an attempt to understand the molecular basis for the lack of inducibility of CYP1A protein expression and activity in Elizabeth River killifish, we analyzed the expression of CYP1A and four other members of the AHR signal transduction pathway: AHR1, AHR2, AHR repressor (AHRR), and AHR nuclear translocator (ARNT). Gene expression was measured by cycle-optimized reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of messenger ribonucleic acid (mRNA) extracted from livers of killifish from the Elizabeth River and King's Creek (VA, USA) (reference site), 36 h after injection with beta-naphthoflavone (BNF, an AHR agonist) or corn oil (carrier control). Hepatic CYP1A mRNA was inducible in King's Creek killifish. However, in Elizabeth River killifish, no induction of CYP1A mRNA was observed, confirming and extending previous results showing no induction of CYP1A protein or catalytic activity in this population. Similarly, AHRR and AHR2 mRNA levels were induced by BNF in King's Creek but not Elizabeth River killifish. No population or treatment-related differences were observed in expression of AHR1 or ARNT mRNAs. The results reveal in Elizabeth River killifish a consistent lack of inducibility of genes that are normally inducible by AHR agonists (CYP1A, AHRR, AHR2). However, the expression of AHR1, AHR2, and AHRR in vehicle-treated fish did not differ between Elizabeth River and King's Creek killifish, suggesting that altered constitutive expression of AHRs or AHRR does not underlie the refractory CYP1A phenotype in Elizabeth River killifish.

Activation of the Ah receptor by extracts of dietary herbal supplements, vegetables, and fruits.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activated by a structurally diverse range of synthetic and natural chemicals, and it mediates the toxic and biological effects of environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The spectrum of chemicals that bind to and activate the AhR signal transduction pathway and the identity of materials containing AhR active chemicals is only now being defined. Utilizing AhR-dependent gel retardation and reporter gene bioassays, the screening of extracts of 22 dietary herbal supplements and 21 food products (vegetables and fruits) was performed to identify those containing AhR agonists. Several herbal extracts (ginseng, Fo-Ti, white oak bark, licorice, ginkgo biloba, and black cohosh) stimulated AhR DNA binding and gene expression to levels between 20 and 60% of that produced by TCDD. Although some food extracts (corn, jalapeño pepper, green bell pepper, apple, Brussels sprout, and potato) were relatively potent activators of AhR DNA binding (30-50% of TCDD), only corn and jalapeño pepper extracts induced AhR-dependent luciferase reporter gene expression. However, dilution of corn, jalapeño pepper, bell pepper, and potato extracts dramatically increased their ability to induce luciferase activity, suggesting that these extracts contained AhR antagonists whose effectiveness was overcome by dilution. Overall, these results demonstrate that dietary products can be a major source of naturally occurring AhR ligands to which animals and humans are chronically exposed.

Relationships among the cell cycle, cell proliferation, and aryl hydrocarbon receptor expression in PLHC-1 cells

Aryl hydrocarbon receptor (AHR) ligands such as 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) cause altered cell proliferation in many tissues in vivo and cell types in vitro, and the AHR has been suggested to play a role in cell cycle regulation in mammalian systems. However, the mechanisms underlying these effects are poorly understood. The overall objective of the present work was to investigate possible interactions between cell proliferation, the cell cycle, and AHR signal transduction in a piscine system, the PLHC-1 cell line, which is being used increasingly in aquatic toxicological research. The specific objectives were to characterize proliferation rates and the cell cycle in these cells, to measure effects of TCDD on cell proliferation, and to determine if expression of the AHR varies during the cell cycle. The doubling time of PLHC-1 cells was determined to be 22 h, and the durations of the G1, S and G2/M stages of the cell cycle were 13, 3, and 6 h, respectively. A minimum seeding density of 1.2×10 5 cells/cm 2 in medium with 10% calf serum and 0.3×10 5 cells/cm 2 in 10% fetal bovine serum was found to be required for subsequent proliferation. Of several cell cycle inhibitors tested, only aphidicolin and nocodazole were effective for obtaining synchronous cell populations. TCDD was found to inhibit PLHC-1 cell proliferation in a time- and dose-dependent manner in multiple passages of one sub-clone, but not in several other sub-clones. Neither AHR mRNA nor protein expression varied during the cell cycle, as measured by RT-PCR and specific binding of [ 3H]TCDD in synchronous PLHC-1 cells. This work establishes techniques for identifying and characterizing possible interactions between the cell cycle and AHR signal transduction in PLHC-1 cells. Taken together, the results indicate that PLHC-1 cells are amenable to analysis of AHR-cell cycle interactions, but that heterogeneity of sub-clones may complicate their use for investigating AHR-mediated changes in proliferation.

Activation of the Ah receptor signaling pathway by prostaglandins.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of a diverse range of chemicals, including the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). Although no endogenous physiological ligand for the AhR has yet been described, numerous studies support the existence of such a ligand(s). Here we have examined the ability of prostaglandins and related chemicals to activate the AhR signaling system. Using two AhR-based bioassay systems we report that relatively high concentrations of several prostaglandins (namely, PGB3, PGD3, PGF3alpha, PGG2, PGH1, and PGH2) can not only stimulate AhR transformation and DNA binding in vitro, but also induce AhR-dependent reporter gene expression in mouse hepatoma cells in culture. PGG2 also induced AhR-dependent reporter gene expression to a level three-to four fold greater than that observed with a maximal inducing dose of TCDD. Sucrose gradient ligand binding analysis revealed that PGG2 could competitively displace [3H]TCDD from the AhR. Overall, our results demonstrate that selected prostaglandins are weak agonists for the AhR and they represent a structurally distinct and novel class of activator of the AhR signal transduction pathway.

Activation of the Ah Receptor Signaling Pathway by Prostaglandins

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of a diverse range of chemicals, including the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). Although no endogenous physiological ligand for the AhR has yet been described, numerous studies support the existence of such a ligand(s). Here we have examined the ability of prostaglandins and related chemicals to activate the AhR signaling system. Using two AhR-based bioassay systems we report that relatively high concentrations of several prostaglandins (namely, PGB3, PGD3, PGF3α, PGG2, PGH1, and PGH2) can not only stimulate AhR transformation and DNA binding in vitro, but also induce AhR-dependent reporter gene expression in mouse hepatoma cells in culture. PGG2 also induced AhR-dependent reporter gene expression to a level three-to four fold greater than that observed with a maximal inducing dose of TCDD. Sucrose gradient ligand binding analysis revealed that PGG2 could competitively displace [3H]TCDD from the AhR. Overall, our results demonstrate that selected prostaglandins are weak agonists for the AhR and they represent a structurally distinct and novel class of activator of the AhR signal transduction pathway. © 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:187–196, 2001