AhR-regulated Genes Research Articles

Role of Nitric Oxide in Downregulation of Cytochrome P450 1a1 and NADPH: Quinone Oxidoreductase 1 by Tumor Necrosis Factor‐α and Lipopolysaccharide

We previously demonstrated that tumor necrosis factor alpha (TNF-alpha) and lipopolysaccharide (LPS) downregulate aryl hydrocarbon receptor (AhR)-regulated genes, such as cytochrome P450 1a1 (Cyp1a1) and NADPH: quinone oxidoreductase 1 (Nqo1) gene expression, yet the mechanisms involved remain unknown. The correlation between the inflammation-mediated suppression of AhR-regulated genes and the TNF-alpha or LPS-induced nitric oxide (NO) production especially in murine hepatoma Hepa 1c1c7 cells has been questioned; therefore we investigated whether NO is involved in the modulation of Cyp1a1 and Nqo1 by TNF-alpha or LPS in Hepa 1c1c7 cells. A significant dose-dependent increase in the inducible nitric oxide synthase (NOS2) expression and NO production were observed by various concentrations of TNF-alpha (1, 5, and 10 ng/mL) and LPS (1 and 5 microg/mL) which was completely inhibited by a NOS2 inhibitor, L-N6-(1-iminoethyl) lysine (L-NIL) (1 mM). Furthermore, TNF-alpha and LPS significantly induced NOS2 expression. Both TNF-alpha and LPS repressed the beta-naphthoflavone (betaNF)-mediated induction of Cyp1a1 and Nqo1 at mRNA and activity levels. The downregulation of Cyp1a1, but not Nqo1, was significantly prevented by L-NIL. However, proxynitrite decomposer, iron tetrakis (N-methyl-4'-pyridyl) porphyrinato (FeTMPyP) (5 microM) did not affect TNF-alpha- and LPS-mediated downregulation of Cyp1a1 and Nqo1 at mRNA and activity levels. These results show that NO, but not peroxynitrite, may be involved in TNF-alpha- and LPS-mediated downregulation of Cyp1a1 without affecting the downregulation of Nqo1.

Molecular and developmental effects of exposure to pyrene in the early life-stages of Cyprinodon variegatus

Polycyclic aromatic hydrocarbons (PAHs) have been connected to developmental toxicity in the early life-stages of many species by their ability to bind to the aryl hydrocarbon receptor (AHR), which dimerizes with ARNT (AHR nuclear translocator) to induce transcription of genes such as CYP1A1. ARNT also dimerizes with HIF (hypoxia-inducible factor α) to induce transcription of genes such as VEGF (vascular endothelial growth factor), suggesting that PAHs may interfere with transcription of VEGF by competing for ARNT. Herein, we address the molecular and developmental effects of exposures to the weak AHR agonist pyrene on the early life-stages of the sheepshead minnow Cyprinodon variegatus. Embryos were exposed under flow-through conditions to 0, 20, 60, or 150 ppb pyrene up to 432 hours post-fertilization (hpf). RNA was extracted at 5 time points (12, 24, 48, 96, and 432 hpf) and changes in CYP1A1 and VEGF expression were assessed by real-time RT-PCR. Since few genes have been documented for the sheepshead minnow, we first cloned and sequenced CYP1A1, VEGF and internal standard 18S rRNA. Pyrene significantly induced the AHR-regulated gene, CYP1A1, in a time- and dose-dependent manner, while pyrene failed to alter the HIF-regulated gene, VEGF. However, VEGF was found to change during various stages of normal development in this study. Although a normal hatch time (5 dpf) was observed for all treatments, pyrene-treated embryos showed dose-dependent abnormalities such as severe dorsal body curvature, mild pericardial and yolk-sac edema, and increased mortality. Taken together, these data indicate that embryonic exposure of sheepshead minnows to pyrene disrupts normal development and alters expression of an AHR/ARNT-regulated gene. In addition, embryonic exposure to pyrene failed to provide evidence of possible AHR-HIF pathway cross-talk since developmental expression of VEGF was unaltered.

Role of AHR2 in the Expression of Novel Cytochrome P450 1 Family Genes, Cell Cycle Genes, and Morphological Defects in Developing Zebra Fish Exposed to 3,3′,4,4′,5-Pentachlorobiphenyl or 2,3,7,8-Tetrachlorodibenzo-p-dioxin

Halogenated agonists for the aryl hydrocarbon receptor (AHR), such as 3,3',4,4',5-pentachlorobiphenyl (PCB126) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), cause developmental toxicity in fish. AHR dependence of these effects is known for TCDD but only presumed for PCB126, and the AHR-regulated genes involved are known only in part. We defined the role of AHR in regulation of four cytochrome P450 1 (CYP1) genes and the effect of PCB126 on cell cycle genes (i.e., PCNA and cyclin E) in zebra fish (Danio rerio) embryos. Basal and PCB126-induced expression of CYP1A, CYP1B1, CYP1C1, and CYP1C2 was examined over time as well as in relation to cell cycle gene expression and morphological effects of PCB126 in developing zebra fish. The four CYP1 genes differed in the time for maximal basal and induced expression, i.e., CYP1B1 peaked within 2 days postfertilization (dpf), the CYP1Cs around hatching (3 dpf), and CYP1A after hatching (14-21 dpf). These results indicate developmental periods when the CYP1s may play physiological roles. PCB126 (0.3-100nM) caused concentration-dependent CYP1 gene induction (EC50: 1.4-2.7nM, Lowest observed effect concentration [LOEC]: 0.3-1nM) and pericardial edema (EC50: 4.4nM, LOEC: 3nM) in 3-dpf embryos. Blockage of AHR2 translation significantly inhibited these effects of PCB126 and TCDD. PCNA gene expression was reduced by PCB126 in a concentration-dependent manner, suggesting that PCB126 could suppress cell proliferation. Our results indicate that the four CYP1 genes examined are regulated by AHR2 and that the effect of PCB126 on morphology in zebra fish embryos is AHR2 dependent. Moreover, the developmental patterns of expression and induction suggest that CYP1 enzymes could function in normal development and in developmental toxicity of PCB126 in fish embryos.

Inducibility of AhR-regulated CYP genes by β-naphthoflavone in the liver, lung, kidney and heart of the pig

The presence and inducibility of CYP enzymes belonging to the family 1 (CYP 1A1, 1A2 and 1B1) and AhR have been studied in liver, lung, kidney and heart of control and beta-naphthoflavone (βNF)-treated pigs. Segments of so far undescribed genes for porcine CYP 1A2, 1B1 and AhR were identified by RT-PCR and their sequences found to be highly homologous to those of the corresponding human genes. The mRNA level of CYP 1A1 was induced by βNF, although to a different extent, in liver, lung, kidney and heart. This transcriptional activation of CYP 1A1 was accompanied in microsomes of all these organs by an induction of 7-ethoxyresorufin deethylase activity (a marker of this isoform) and an increase in a protein band immunoreactive with anti-rat CYP 1A1. An increase in CYP 1A2 transcription and in activity of microsomal 7-methoxyresorufin demethylase and acetanilide 4-hydroxylase (both markers of 1A2) was observed in the liver and, to a very small extent, in the lung but not in kidney and heart. As to CYP 1B1, its transcription was detected in liver, lung and heart only following the βNF treatment; however this mRNA expression did result in any detectable microsomal 17β-estradiol 4-hydroxylase activity (a marker of this isoform). The CYPs induced by βNF were further investigated by using some other marker activities. It was found that porcine CYP 1A1 and 1A2, unlike the human counterparts, could only deethylate 7-ethoxycomarin to a very small extent, if at all, whereas 7-ethoxy 4-trifluoromethylcoumarin was a good substrate for pig CYP 1A1. Overall, our results demonstrated a differential expression and regulation of the AhR-mediated CYP genes in liver, lung, kidney and heart of the pig.naphthoflavone

Induction of Cyp1a1 Is a Nonspecific Biomarker of Aryl Hydrocarbon Receptor Activation: Results of Large Scale Screening of Pharmaceuticals and Toxicants in Vivo and in Vitro

Expression of Cyp1a1 and its related enzyme activity have long been used as a biomarker for aryl hydrocarbon receptor (AhR) activation and a warning of dioxin-like toxicity. As a result, induction of Cyp1a1 by pharmaceutical drug candidates or environmental contaminants raises significant concern in risk assessment. The current study evaluates the specificity of Cyp1a1 induction as a marker for AhR affinity and activation and provides context to assess the relevancy of AhR activation to risk assessment. In vivo experiments examined the expression of Cyp1a1 and other AhR-regulated genes in liver, kidney, and heart in response to 596 compounds. From this data set, a subset of 147 compounds was then evaluated for their ability to activate or bind to the AhR using a combination of gel shift, reporter gene, and competitive receptor binding assays. Whereas in vivo Cyp1a1 mRNA expression is a sensitive marker for AhR activation, it lacks specificity, because 81 (59%) of 137 compounds were found to significantly induce Cyp1a1 in vivo but were not verified to bind or activate the AhR in vitro. Combining in vivo and in vitro findings, we identified nine AhR agonists, six of which are marketed therapeutics and have been approved by the U.S. Food and Drug Administration, including leflunomide, flutamide, and nimodipine. These drugs do not produce dioxin-like toxicity in rats or in humans. These data demonstrate that induction of Cyp1a1 is a nonspecific biomarker of direct AhR affinity and activation and lend further support to the hypothesis that Cyp1a1 induction and/or AhR activation is not synonymous with dioxin-like toxicity.

β-Naphthoflavone and 3′-methoxy-4′-nitroflavone exert ambiguous effects on Ah receptor-dependent cell proliferation and gene expression in rat liver ‘stem-like’ cells

Both natural and synthetic flavonoids are known to interact with the aryl hydrocarbon receptor (AhR); however, their agonist/antagonist properties in vitro have been so far studied mostly in the context of cytochrome P450 1A1 gene ( Cyp1a1) regulation. We investigated effects of two synthetic flavones known either as AhR agonist (β-naphthoflavone; BNF) or antagonist (3′-methoxy-4′-nitroflavone; 3M4NF), using an in vitro model of liver ‘stem-like’ cells, on expression of various AhR target genes and AhR-dependent cell proliferation. We found that the presumed antagonist 3M4NF induces a partial nuclear translocation and activation of AhR. Although inhibiting the 2,3,7,8-tetrachlorodibenzo- p-dioxin-induced Cyp1a1 expression, 3M4NF alone induced a minor increase of CYP1A1 mRNA and protein. However, 3M4NF did not induce AhR binding to synthetic dioxin response elements (DRE). In contrast to Cyp1a1, 3M4NF induced a marked expression of other AhR-regulated genes, such as Cyp1b1 and Nqo1, as well as transcriptional repression of Cdh13 gene, confirming that its effects may be promoter-context specific. Like BNF, 3M4NF induced AhR-dependent cell proliferation of contact-inhibited rat liver ‘stem-like’ WB-F344 cells, associated with a marked upregulation of Cyclin A, as well as the downregulation of proteins involved in formation of cell–cell contacts. Based on these experimental findings, we conclude that partial agonists/antagonists of AhR can increase cell proliferation rate and AhR-dependent genes expression in both cell type- and gene-specific manner. The specificity of effects of flavones on diverse AhR targets should be taken into account, when studying AhR signaling using presumed AhR antagonists.

D14 Inducibility of AHR‐regulated genes by β‐naphtoflavone in the liver, lung and heart of the pig

D14 Inducibility of AHR‐regulated genes by <i>β</i>‐naphtoflavone in the liver, lung and heart of the pig

Aryl Hydrocarbon Receptor- and Calcium-dependent Induction of the Chemokine CCL1 by the Environmental Contaminant Benzo[a]pyrene

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed immunotoxic environmental contaminants well known to regulate expression of pro-inflammatory cytokines such as interleukine-1beta and tumor necrosis factor-alpha. In the present study, we demonstrated that the chemokine CCL1, notably involved in cardiovascular diseases and inflammatory or allergic processes, constitutes a new molecular target for PAHs. Indeed, exposure to PAHs such as benzo[a]pyrene (BP) markedly increased mRNA expression and secretion of CCL1 in primary human macrophage cultures. Moreover, intranasal administration of BP to mice enhanced mRNA levels of TCA3, the mouse orthologue of CCL1, in lung. CCL1 induction in cultured human macrophages was fully prevented by targeting the aryl hydrocarbon receptor (AhR) through chemical inhibition or small interfering RNA-mediated down-modulation of its expression. In addition, BP and the potent AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin were found to enhance activity of a CCL1 promoter sequence containing a consensus xenobiotic-responsive element known to specifically interact with AhR. Moreover, 2,3,7,8-tetrachlorodibenzo-p-dioxin triggered AhR binding to this CCL1 promoter element as revealed by chromatin immunoprecipitation experiments and electrophoretic mobility shift assays. In an attempt to further characterize the mechanism of CCL1 induction, we demonstrated that BP was able to induce an early and transient increase of intracellular calcium concentration in human macrophages. Inhibition of this calcium increase, using the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester or the calcium store-operated channel inhibitor 2-aminoethoxydiphenyl borate, fully blocked CCL1 up-regulation. Taken together, these results bring the first demonstration that PAHs induce expression of the chemokine CCL1 in an AhR- and calcium-dependent manner.

Role of GAC63 in Transcriptional Activation Mediated by the Aryl Hydrocarbon Receptor

The aryl hydrocarbon receptor (AHR), a member of the basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) gene family, binds a variety of polycyclic aromatic hydrocarbons and mediates their toxic effects. GAC63 has been shown to act as a coactivator in nuclear receptor-mediated gene transcription. In this report, we demonstrate that GAC63 interacts with AHR through its bHLH-PAS domain. Overexpression of GAC63 greatly enhanced AHR-regulated reporter gene activity in a ligand-dependent manner in transient transfection assays. Upon ligand treatment, endogenous GAC63 was recruited to the xenobiotic response element of the mouse CYP1A1 gene, an AHR-responsive gene. Reduction of the endogenous GAC63 level by small interfering RNA inhibited transcriptional activation by AHR. These findings reveal a new function of GAC63 in AHR-mediated gene transcription.

Tissue distribution and function of the Aryl hydrocarbon receptor repressor (AhRR) in C57BL/6 and Aryl hydrocarbon receptor deficient mice

The Aryl hydrocarbon receptor repressor (AhRR) is a new member of bHLH-PAS proteins which is important in the regulation of cell growth and differentiation. The AhRR shares structural similarities with Aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT). The AhRR is thought to be involved in transcriptional control of AhR-regulated genes by sequestering ARNT. Most of the knowledge of regulation and function of the AhRR is from studies in cell lines. Here, we report the tissue distribution of AhRR in AhR deficient and wild type C57BL/6 mice. In addition, the inducibility of the AhRR and Cytochrome P450 (CYP) 1A1 in response to benzo(a)pyrene (B(a)P) (10 mg/kg bw i.p.) was investigated. The results show that the AhRR mRNA expression pattern in untreated C57BL/6 mice varies across tissues with high levels in hearts and brains. In other tissues, AhRR mRNA expression was low. In contrast to wild-type animals, the tissue levels in AhR-/- mice were about two to three orders of magnitude lower. Treatment of wild-type animals with B(a)P resulted in an induced AhRR expression in liver, spleen, lung and ovary. No significant induction of AhRR mRNA was found in brain and heart tissues, which have a constitutively high level of AhRR expression. Simultaneous measurements of CYP1A1 and AhRR mRNA expression do not strongly support the view that the AhRR tissue pattern triggers the tissue specific responsiveness of AhR-regulated genes to B(a)P treatment.

Constitutive Activation and Environmental Chemical Induction of the Aryl Hydrocarbon Receptor/Transcription Factor in Activated Human B Lymphocytes

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates immunosuppression induced by a variety of ubiquitous environmental pollutants, including polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and dioxins. Although the normal physiological role for the AhR in the absence of environmental chemicals is uncertain, recent studies suggest its contribution to cell growth and apoptosis. Because B cells seem to be directly affected by AhR ligands in animal models, it was postulated that the AhR is predominantly expressed in activated human B cells and that it may contribute to cell growth regulation. To begin to address these issues and to extend detailed analyses of AhR function to a human system, AhR expression in resting and activated human B cells was studied. In addition, the response of activated B cells to an environmental AhR ligand was investigated to provide insight into a possible physiological role for the AhR. Resting peripheral human B cells expressed little or no AhR. However, activation with CpG or CD40 ligand profoundly up-regulated AhR mRNA and protein. AhR nuclear translocation, constitutive DNA binding, and induction of an AhR-regulated gene, CYP1A1, in stimulated B cells in the absence of exogenous ligands suggested constitutive AhR activation. Cell division was not required for AhR up-regulation. Treatment of AhR-expressing B cells with a prototypic environmental AhR ligand, benzo[a]pyrene, significantly suppressed cell growth. These data help explain the sensitivity of B cells to environmental AhR ligands and strongly suggest that the AhR plays an important function within the human B cell compartment.

Regulation of theCyp2a5Gene Involves an Aryl Hydrocarbon Receptor-Dependent Pathway

We have investigated the role of the aryl hydrocarbon receptor (AHR) in the regulation of the Cyp2a5 gene. The C57BL/6 and DBA/2 mouse strains with a genetically determined difference in AHR function were used to study the CYP2A5 induction by typical AHR ligands, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene. The CYP2A5 mRNA up-regulation in these mouse strains showed a difference in response, typical for AHR-regulated genes, both by TCDD in cultured primary hepatocytes and by 3-methylcholanthrene in vivo. In primary hepatocytes, TCDD caused a 3-fold elevation of the CYP2A5 protein level and a similar induction of the CYP2A5-catalyzed coumarin 7-hydroxylation activity. In reporter gene assays, the Cyp2a5 promoter region -3033 to +10 mediated a 2- to 5-fold induction of luciferase activity by TCDD treatment in primary hepatocytes and in Hepa-1 hepatoma cells with an intact AHR/AHR nuclear translocator (ARNT) complex. In Hepa-1 variant cell lines with deficiencies in the AHR/ARNT complex, the absence of ARNT abolished the induction. A putative AHR response element (XRE) was identified in the Cyp2a5 promoter at the position -2514 to -2492 and found to interact with the AHR/ARNT heterodimer. Transfection experiments combined with mutation of the XRE site indicated that the site partly mediates the TCDD induction of Cyp2a5. An additional AHR-dependent mechanism also regulates the proximal promoter of the Cyp2a5 gene. In conclusion, our studies showed that AHR ligands up-regulate Cyp2a5 transcriptionally by an AHR/ARNT-dependent mechanism and established Cyp2a5 as a novel AHR-regulated gene.

Metabolic fate of the Ah receptor ligand 6-formylindolo[3,2- b]carbazole

The physiological role of the aryl hydrocarbon receptor (AhR), a member of the basic helix–loop–helix PER-ARNT-SIM (PAS) transcription factor family is not known. We have suggested that the AhR is involved in light signaling through binding of photoproducts with high AhR affinity. This suggestion is based on (i) the high AhR affinity of the tryptophan photoproduct formylindolo[3,2- b]carbazole (FICZ), (ii) the induction of rapid and transient expression of AhR-regulated genes by FICZ and by extracts of UV-irradiated tryptophan as well as (iii) the fact that light induces the AhR-regulated cytochrome P450s CYP1A1, CYP1B1 and CYP2S1. The transient mRNA expression caused by light and tryptophan photoproducts suggests that the biotransformation enzymes induced by AhR activation take part in a metabolic degradation of the natural AhR ligand. This study aimed at identifying the involvement of phase I and phase II enzymes in the metabolic degradation of FICZ. A cytochrome P450-dependent metabolism of FICZ giving rise to preferentially mono- and di-hydroxylated derivatives has earlier been reported. In the present study, rat and human hepatic S9 mixes were employed together with specific enzyme inhibitors and cofactors. Compared to the Aroclor-induced rat liver S9, the non-induced rat liver S9 and the human liver S9 caused a more complex metabolite profile of FICZ. The CYP1A1 enzyme was confirmed to be the most important enzyme for the first step in the metabolism. CYP1A2 was found to have overlapping specificity with CYP1A1 being able to form the same major metabolites although with different kinetics. CYP1B1 turned out to be preferentially involved in the further metabolism of dihydroxylated metabolites. Microsomal epoxide hydrolase, and as yet not identified forms of sulphotransferases and glucuronosyltransferases were also found to take part in the metabolic degradation of FICZ. Thus, tryptophan photoproducts fit into a model in which the ligand-activated AhR signaling is autoregulated by the induced metabolic enzymes.

Subchronic Exposure to TCDD, PeCDF, PCB126, and PCB153: Effect on Hepatic Gene Expression

We employed DNA microarray to identify unique hepatic gene expression patterns associated with subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other halogenated aromatic hydrocarbons (HAHs). Female Harlan Sprague-Dawley rats were exposed for 13 weeks to toxicologically equivalent doses of four different HAHs based on the toxic equivalency factor of each chemical: TCDD (100 ng/kg/day), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF; 200 ng/kg/day), 3,3′,4,4′,5-pentachlorobiphenyl (PCB126; 1,000 ng/kg/day), or 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153; 1,000 μg/kg/day). Global gene expression profiles for each exposure, which account for 8,799 gene probe sets contained on Affymetrix RGU34A GeneChips, were compared by principal components analysis. The aryl hydrocarbon receptor (AhR) ligands TCDD, PeCDF, and PCB126 produced very similar global gene expression profiles that were unique from the nonAhR ligand PCB153, underscoring the extensive impact of AhR activation and/or the resulting hepatic injury on global gene expression in female rat liver. Many genes were co-expressed during the 13-week TCDD, PeCDF, or PCB126 exposures, including classical AhR-regulated genes and some genes not previously characterized as being AhR regulated, such as carcinoembryonic-cell adhesion molecule 4 (C-CAM4) and adenylate cyclase-associated protein 2 (CAP2). Real-time reverse-transcriptase polymerase chain reaction confirmed the increased expression of these genes in TCDD-, PeCDF-, and PCB126-exposed rats as well as the up- or down-regulation of several other novel dioxin-responsive genes. In summary, DNA microarray successfully identified dioxin-responsive genes expressed after exposure to AhR ligands (TCDD, PeCDF, PCB126) but not after exposure to the non-AhR ligand PCB153. Together, these findings may help to elucidate some of the fundamental features of dioxin toxicity and may further clarify the biologic role of the AhR signaling pathway.

Modulation of aryl hydrocarbon receptor-regulated gene expression by arsenite, cadmium, and chromium.

Aryl hydrocarbon receptor (AhR) ligands and heavy metals are environmental co-contaminants and their molecular interaction may disrupt the coordinated regulation of AhR-dependent phase I and II drug metabolizing enzymes. To determine the effect of heavy metals on the AhR-regulated genes: cytochrome P4501A1 (Cyp1a1), NAD(P)H: quinone oxidoreductase (QOR) and glutathione S-transferase Ya (GST Ya), murine hepatoma Hepa 1c1c7 cells were treated with increasing concentrations of As3+ (1-10 microM), Cd2+ (1-25 microM) and Cr6+ (1-25 microM) with or without the AhR ligands: 2,3,7,8-tetrachlorodibenzo-p-dioxin (0.1 nM), 3-methylcholanthrene (0.25 microM), beta-naphthoflavone (10 uM), or benzo[a]pyrene (1 microM). Our results show that AhR ligands alone and As3+ or Cd2+ alone increased the catalytic activities and mRNA levels of all AhR-regulated genes. When metals were co-administered with an AhR ligand, all three metals inhibited the induction of Cyp1a1 activity by the AhR ligands but potentiated its mRNA and protein expression. In addition, all metals enhanced QOR and GST Ya at the activity and mRNA levels but modulated their induction by AhR ligands in a concentration, metal, and AhR ligand-dependent manner. Generally, Cr6+ inhibited while As3+ and Cd2+ potentiated the induction of QOR and GST Ya activities and mRNA levels. The three metals enhanced the expression of heme oxygenase-1, which coincided with the changes in the phase I and phase II enzyme activities. These results show that the ability of metals to alter the capacity of AhR ligands to induce the bioactivating phase I and the detoxifying phase II enzymes will influence the carcinogenicity and mutagenicity of the AhR ligands.

Differential effects of mercury, lead and copper on the constitutive and inducible expression of aryl hydrocarbon receptor (AHR)-regulated genes in cultured hepatoma Hepa 1c1c7 cells.

Both simultaneous and sequential exposure to heavy metals and aryl hydrocarbon receptor (AHR)-ligands potentially occur in human populations, yet there have been relatively few studies of combined effects of heavy metals and AHR-ligands on AHR-regulated genes. To investigate the effects of heavy metals on AHR-regulated genes; cytochrome P450 1a1 (cyp1a1), NAD(P)H:quinone oxidoreductase (QOR) and glutathione S-transferase Ya (GST Ya), murine hepatoma Hepa 1c1c7 cells were incubated with increasing concentrations of Hg2+ (2.5-10 microM), Pb2+ (10-100 microM), and Cu2+ (1-100 microM) alone or with the AHR-ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (0.1 nM), 3-methylcholanthrene (0.25 microM), beta-naphthoflavone (10 microM), or benzo[a]pyrene (1 microM). The results clearly showed that metals alone did not significantly alter the cyp1a1 activity and protein levels but increased its mRNA expression, whereas a significant reduction in AHR ligand-mediated induction of cyp1a1 activity was observed by all metals. The decrease in cyp1a1 activity was associated with an increase, no change, or decrease in cyp1a1 mRNA and protein levels by Hg2+, Pb2+ and Cu2+ respectively, suggesting pre- and post-transcription mechanisms are involved. With respect to QOR, the activity and mRNA levels were increased by all metals in the absence or presence of an AHR-ligand, with the exception of Cu2+ which significantly decreased the induction of QOR. Differently, GST Ya activity was significantly increased by Cu2+ and Pb2+ and inhibited by Hg2+, while its mRNA was increased by Hg2+ and Pb2+ and decreased by Cu2+. All metals significantly increased the expression of heme oxygenase-1, which coincided with the changes in the phase I and phase II enzyme activities. These results demonstrate that heavy metals differentially modulate the constitutive and the inducible expression of AHR-regulated genes.

Overexpression of latent transforming growth factor-beta binding protein 1 (LTBP-1) in dioxin receptor-null mouse embryo fibroblasts.

The aryl hydrocarbon receptor (AhR) is a transcriptional regulator of genes involved in xenobiotic metabolism. Increasingly clear is also the role of the AhR in the control of cell growth and proliferation. By analyzing differential patterns of gene expression between wild-type (AhR+/+) and null (AhR-/-) mouse embryo fibroblasts (MEF), we have identified latent transforming growth factor-beta binding protein 1 (LTBP-1) as a negatively AhR-regulated gene in the absence of xenobiotics. Ltbp-1 mRNA and protein expression were markedly increased in AhR-/- MEF. Furthermore, secreted LTBP-1 was elevated in the culture medium and the extracellular matrix of AhR-null MEF. Actinomycin D inhibited Ltbp-1 mRNA overexpression, suggesting regulation at the transcriptional level. AhR activation by dioxin (TCDD) downregulated Ltbp-1, again suggesting an AhR-regulated mechanism. Treatment of AhR+/+ MEF with transforming growth factor-beta(TGF-beta) downregulated AhR and, simultaneously, increased Ltbp-1, further supporting the role of this receptor in LTBP-1 expression. AhR-/- conditioned medium had higher levels of active and total TGF-beta activity, suggesting a role for LTBP-1 in maintaining extracellular TGF-beta concentrations. TGF-beta did not appear to directly regulate Ltbp-1 given that addition of TGFbeta neutralizing antibody or TGFbeta protein to AhR-/- MEF had no effect on Ltbp-1 expression. AhR-/- MEF had lower levels of matrix metalloproteinase 2 (MMP-2) activity, which could not be attributable to MMP-2 mRNA downregulation or MMP-inhibitors Timp-1 and Timp-2 overexpression. These data identify LTBP-1 as one of the few AhR-regulated genes not involved in xenobiotic metabolism and also support the implication of the AhR in controlling TGFbeta activity and cell proliferation.

Establishment of a Rat Hepatic Cell Line, KanR2-XL8, for a Reporter Gene Assay of Aryl Hydrocarbon Receptor Ligands

To establish a highly sensitive and convenient reporter gene assay for aryl hydrocarbon receptor (AhR) ligands, the chimera plasmid xenobiotic responsible element-luciferase (XRE-Luc) containing an XRE, minimal SV40 promoter, and luciferase reporter gene, was first constructed. The XRE-Luc and the expression vector pRC/CMV containing a neomycin-resistant gene were transfected into a rat hepatic cell line, Kan-R2, and then KanR2-XL8 was selected as a cell clone, which showed the highest response to the induction of luciferase by aryl hydrocarbons, 3-methylcholanthlene (3-MC) and benzo[a]pyrene. Furthermore, AhR-regulated genes, CYP1A1 and CYP1B1, in KanR2-XL8 cells were also activated by 3-MC. In the present study, we established a hepatic cell line, KanR2-XL8, that is useful for screening of AhR ligands with two parameters, the activations of the transfected luciferase gene and the AhR-regulated genes in a host cell.

TCDD-induced expression of Ah receptor responsive genes in the pituitary and brain of cellular retinol-binding protein (CRBP-I) knockout mice

The 2,3,7,8-tetrachlorodibenzo- p-dioxin (2,3,7,8-TCDD) is a highly toxic, widespread environmental contaminant. Most of the toxic damage caused by TCDD is considered to be secondary to the binding of TCDD to the aryl hydrocarbon receptor, the AH receptor (AHR). TCDD is known to affect the vitamin A homeostasis. The effects of TCDD on body weight and on the expressions of AHR-associated genes may be modulated by the retinoid system. Therefore, we investigated the regulation pattern of expression of the AHR associated genes after TCDD exposure in wild-type 129/SV/C57BL/6 mice and in mice deficient in cellular retinol-binding protein type I (CRBP-I) of the same mixed genetic background. A single oral dose of TCDD (50 or 250 μg/kg) was administrated by gavage. Different brain areas, including cortex, hypothalamus, cerebellum, and olfactory bulb, as well as pituitary and liver, were dissected from CRBP-I knockout homozygous mice (−/−) and wild-type mice (+/+) killed at two time points (7 or 28 days after treatment). Compared with vehicle-treated controls, the relative levels of cytochrome P450 1A1 (CYP1A1) mRNA and protein expression in TCDD-treated animals were dramatically increased in mice of both CRBP-I genotypes (−/−, +/+). CYP1A1 mRNA levels increased up to 1400-fold in the pituitary, 110-fold in the brain, and up to 1600-fold in the liver. A general observation was that the relative induction of CYP1A1 and AHR transcription after TCDD dosing was highest in the +/+ mice. A high TCDD-induced aryl hydrocarbon receptor repressor (AHRR) mRNA expression was observed in the liver and in brain tissues. Interestingly, however, mice that lacked the CRBP-I protein (−/−) were found to have a significantly higher basal expression of AHRR gene in the pituitary compared to the wild-type (CRBP-I +/+) mice and in accordance a less pronounced induction of CYP1A1 and AHR was observed in the pituitary of the −/− mice. Immunohistochemical double staining analyses of the expression pattern of CYP1A1 and AHRR proteins in the pituitary revealed a colocalization of these two proteins. We conclude that the vitamin A homeostasis seems to have some influence to the TCDD-induced activation of AHR-regulated gene transcription in the brain and pituitary of the adult mouse.

Characterization of in Vitro Metabolites of the Aryl Hydrocarbon Receptor Ligand 6-Formylindolo[3,2-b]carbazole by Liquid Chromatography-Mass Spectrometry and NMR.

The tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) exhibits the highest aryl hydrocarbon receptor (AhR) binding affinity reported so far. In different cells, in vitro, both extracts of UV-irradiated tryptophan and the synthesized pure compound FICZ induce a rapid and transient expression of AhR-regulated genes. The transient induction suggests that the biotransformation gene battery induced by AhR activation takes part in a metabolic degradation of the ligand, whereby a low steady-state level is regained. The down-regulation of AhR-regulated gene expression was previously shown to be dependent on cytochrome P450 1A1 (CYP1A1). Metabolism of FICZ generates five major metabolites, which appeared as three peaks (M1-M3) in the high performance liquid chromatography. The aim of the present study was to use rat liver S9 from Aroclor-pretreated rats to produce large enough quantities of FICZ metabolites for structure characterization and to determine their product precursor relationship. NMR analysis of large combined fractions of the metabolites indicated that M3 and M2 contained 2 isomers, respectively. By means of liquid chromatography-mass spectrometry (negative ion electrospray mode) and NMR spectroscopy (by (1)H-NMR, correlation spectroscopy, and nuclear Overhauser effect spectroscopy techniques) five metabolites of FICZ were identified, and their structures were elucidated. The molecular weights of the two M3 isomers were 300 and both M2 and M1 compounds demonstrated molecular weights of 316, corresponding to addition of one (M3) and of two oxygen (M2 and M1), respectively. The structures were assigned as 2- and 8-hydroxy (M3), 2,10- and 4,8-dihydroxy (M2) and 2,8-dihydroxy derivatives of indolo[3,2-b]carbazole-6-carboxaldehyde (6-formylindolo[3,2-b]carbazole).