The aryl hydrocarbon receptor is a modulator of anti-viral immunity

Although immune modulation by AhR ligands has been studied for many years, the impact of AhR activation on host defenses against viral infection has not, until recently, garnered much attention. The development of novel reagents and model systems, new information regarding anti-viral immunity, and a growing appreciation for the global health threat posed by viruses have invigorated interest in understanding how environmental signals affect susceptibility to and pathological consequences of viral infection. Using influenza A virus as a model of respiratory viral infection, recent studies show that AhR activation cues signaling events in both leukocytes and non-immune cells. Functional alterations include suppressed lymphocyte responses and increased inflammation in the infected lung. AhR-mediated events within and extrinsic to hematopoietic cells has been investigated using bone marrow chimeras, which show that AhR alters different elements of the immune response by affecting different tissue targets. In particular, suppressed CD8⁺ T cell responses are due to deregulated events within leukocytes themselves, whereas increased neutrophil recruitment to and IFN-γ levels in the lung result from AhR-regulated events extrinsic to bone marrow-derived cells. This latter discovery suggests that epithelial and endothelial cells are overlooked targets of AhR-mediated changes in immune function. Further support that AhR influences host cell responses to viral infection are provided by several studies demonstrating that AhR interacts directly with viral proteins and affects viral latency. While AhR clearly modulates host responses to viral infection, we still have much to understand about the complex interactions between immune cells, viruses, and the host environment.     

Investigating the role of the aryl hydrocarbon receptor in benzene-initiated toxicity in vitro

Chronic occupational exposure to benzene has been correlated with aplastic aneamia and acute myelogenous leukemia, however mechanisms behind benzene toxicity remain unknown. Interestingly, benzene-initiated hematotoxicity is absent in mice lacking the aryl hydrocarbon receptor (AhR) suggesting an imperative role for this receptor in benzene toxicities. This study investigated two potential roles for the AhR in benzene toxicity using hepa 1c1c7 wild type and AhR deficient cells. Considering that many toxic effects of AhR ligands are dependent on AhR activation, our first objective was to determine if benzene, hydroquinone (HQ) or benzoquinone (BQ) could activate the AhR. Secondly, because the AhR regulates a number of enzymes involved in oxidative stress pathways, we sought to determine if the AhR had a role in HQ and BQ induced production of reactive oxygen species (ROS). Dual luciferase assays measuring dioxin response element (DRE) activation showed no significant change in DRE activity after exposure to benzene, HQ or BQ for 24h. Immunofluorescence staining showed cytosolic localization of the AhR after 2h incubations with benzene, HQ or BQ. Western blot analysis of cells exposed to benzene, HQ or BQ for 1, 12 and 24h did not demonstrate induction of CYP1A1 protein expression. Dichlorodihydrofluorescein staining of cells exposed to benzene, HQ or BQ revealed that the presence of the AhR did not affect BQ and HQ induced ROS production. These results indicate that the involvement of the AhR in benzene toxicity does not seem to be through classical activation of this receptor or through interference of oxidative stress pathways.     

The aryl hydrocarbon receptor is required for normal gonadotropin responsiveness in the mouse ovary

The aryl hydrocarbon receptor (AHR) mediates the toxicity of a variety of environmental chemicals. Although little is known about the physiological role of the AHR, studies suggest that it plays an important role in regulating ovulation because Ahr deficient (AhRKO) mice have a reduced number of ovulations compared to wild-type (WT) mice. The reasons for the reduced ability of AhRKO mice to ovulate are unknown. Normal ovulation, however, requires estrous cyclicity, appropriate luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels, and LH and FSH responsiveness. Thus, the purpose of this study was to test the hypothesis that Ahr deletion regulates ovulation by altering cyclicity, FSH and LH levels, follicle-stimulating hormone receptor (Fshr) and luteinizing hormone receptor (Lhcgr) levels and/or gonadotropin responsiveness. The data indicate that AhRKO and WT mice have similar levels of FSH and LH, but AhRKO mice have reduced Fshr and Lhcgr mRNA levels compared to WT mice. Furthermore, AhRKO ovaries contain fewer corpora lutea compared to WT ovaries after 5 IU equine chorionic gonadotropin (eCG) treatment. Lastly, both AhRKO and WT mice ovulate a similar number of eggs in response to 5 IU human chorionic gonadotropin (hCG), but AhRKO mice ovulate fewer eggs than WT mice in response to 2.5 IU and 1.25 IU hCG. Collectively, these data indicate that AhRKO follicles have a reduced capacity to ovulate compared to WT follicles and that this is due to reduced responsiveness to gonadotropins. Thus, in addition to mediating toxicity of environmental chemicals, the Ahr is required for normal ovulation.     

Physiological role of the aryl hydrocarbon receptor in mouse ovary development.

The aryl hydrocarbon receptor (AhR) regulates the toxicity of environmental contaminants such as 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). As the physiological role of the AhR in the ovary is unknown, the purpose of this study was to test the hypothesis that the AhR regulates the appearance and numbers of ovarian follicles. Ovaries were harvested from AhR-deficient (AhRKO) and wild-type mice on gestational day 18 (GD 18) and postnatal days (PND) 2-3, 8, 32-35, and 53. Complete serial sections of ovaries were evaluated histologically for the presence of germ cells and follicles. On GD 18, there was no difference in the number of germ cells per ovary between AhRKO and wild-type fetuses. However, by PND 2-3, AhRKO mice had significantly more fully formed primordial follicles (AhRKO = 38,440 +/- 3632 versus wild-type = 21,120 +/- 2688) and fewer single germ cells than wild-type mice (AhRKO = 12,696 +/- 1192 vs. wild-type = 18,160 +/- 720). On PND 8 and 32-35, there was no difference in the number of follicles between AhRKO and wild-type mice but by PND 53, AhRKO mice had significantly fewer antral follicles than wild-type (AhRKO = 3416 +/- 480 vs. wild-type = 6776 +/- 1024). Taken together, these results suggest that the AhR may play a role in the formation of primordial follicles and the regulation of antral follicle numbers.     

3',4'-dimethoxyflavone as an aryl hydrocarbon receptor antagonist in human breast cancer cells.

Treatment of MCF-7 and T47D human breast cancer cells with 3', 4'-dimethoxyflavone (3',4'-DMF) alone did not induce CYP1A1-dependent ethoxyresorufin O:-deethylase (EROD) activity or reporter gene activity in cells transfected with an aryl hydrocarbon (Ah)-responsive construct (pRNH11c). In contrast, 1 nM 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) induced up to a 50- to 80-fold increase in EROD and reporter gene activity in MCF-7 and T47D cells. In cells cotreated with 1 nM TCDD plus 0.1-10 microM 3',4'-DMF, there was a concentration-dependent decrease in the TCDD-induced responses, with 100% inhibition observed at the 10 microM concentration. Gel mobility shift assays using rat liver cytosol and breast cancer cell nuclear extracts showed that 3',4'-DMF alone did not transform the AhR to its nuclear binding form, but inhibited TCDD-induced AhR transformation in rat liver cytosol and blocked TCDD-induced formation of the nuclear AhR complex in MCF-7 and T47D cells. TCDD also inhibited estrogen-induced transactivation in MCF-7 cells, and this response was also blocked by 3',4'-DMF, confirming the AhR antagonist activity of this compound in breast cancer cells.     

A new cross-talk between the aryl hydrocarbon receptor and RelB, a member of the NF-κB family

The discovery of the new crosstalk between the aryl hydrocarbon receptor (AhR) and the NF-κB subunit RelB may extend our understanding of the biological functions of the AhR and at the same time raises a number of questions, which will be addressed in this review. The characteristics of this interaction differ from that of AhR with RelA in that the latter appears to be mostly negative unlike the collaborative interactions of AhR/RelB. The AhR/RelB dimer is capable of binding to DNA response elements including the dioxin response element (DRE) as well as NF-κB binding sites supporting the activation of target genes of the AhR as well as NF-κB pathway. Further studies show that AhR/RelB complexes can be found not only in lymphoid cells but also in a human hepatoma cell line (HepG2) or breast cancer cell line (MDA-MB-231). RelB has been implicated in carcinogenesis of breast cancer for instance and RelB is known to be a critical factor for the function and differentiation of dendritic cells; interestingly the participation of AhR in both processes has been suggested recently, which offers the great potential to expand the scope of the physiological roles of the AhR. There is evidence indicating that RelB may serve as a pro-survival factor, including its ability to promote “inflammation resolution” besides the association of RelB with inflammatory disorders. Based on such information, a hypothesis has been proposed in this review that AhR together with RelB functions as a coordinator of inflammatory responses.     

Species difference in the regulation of cytochrome P450 2S1: lack of induction in rats by the aryl hydrocarbon receptor agonist PCB126

1. CYP2S1 is an evolutionarily conserved, mainly extra-hepatic member of the CYP2 family and proposed to be regulated by the aryl hydrocarbon receptor (AhR).

2. The present study explores AhR’s regulation of CYP2S1 in male Sprague Dawley rats using PCB126 (3,3′,4,4′,5-pentachlorobiphenyl), the most potent AhR agonist among the PCBs. Additionally, CYP2S1 expression was examined after treatments with the classic CYP-inducers β-naphthoflavone (β-NF, AhR activator), phenobarbital (PB, CAR activator) and dexamethasone (Dex, PXR activator). CYP2S1 and CYP1A1/2, CYP1B1, CYP2B and CYP3A mRNAs were measured in liver, lung, spleen, stomach, kidney, and thymus at different time points.

3. Constitutive CYP2S1 was expressed at comparable levels to other CYPs with the highest expression levels in stomach, kidney and lung. CYP2S1 mRNA was only non-significantly elevated by β-NF in liver tissues. PCB126 did not increase CYP2S1 mRNA in any organ and at any time point examined despite a significant induction of CYP1 genes. PCB126 reduced CYP2S1 mRNA by 40% (not significant) from the 7th post-exposure day in thymus. PB and Dex had no effect on CYP2S1 mRNA levels.

4. These observations show that in this model CYP2S1 is not, or only weakly, regulated by AhR and not induced by CAR or PXR activators.

     

Modulation of aryl hydrocarbon receptor regulated genes by acute administration of trimethylarsine oxide in the lung,kidney and heart of C57BL/6 mice

Abstract

1.?Arsenite alters the expression of aryl hydrocarbon receptor (AhR)-regulated genes in extrahepatic tissues; yet, the effect of organic arsenicals still unknown. Therefore, C57BL/6 mice received trimethylarsine oxide (TMAO; 13?mg/kg i.p.) with or without 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 15?μg/kg), and euthanized at 6 or 24?h.

2.?Our results demonstrated that TMAO increased Cyp1a1 and Cyp1b1 mRNA, protein and activity in the lung. TMAO potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1a2 mRNA, protein and activity in the lung. In the kidney, TMAO increased Cyp1b1 mRNA and protein. TMAO potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1b1 mRNA, protein and activity. In the heart, TMAO potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1b1 mRNA.

3.?Moreover, TMAO induced Nqo1 mRNA in the lung, kidney and heart, with subsequent increase in Nqo1 protein and activity in the lung. TMAO increased Gsta mRNA in the heart; and increased Gsta protein and activity in the lung and kidney. TMAO increased Nqo1 mRNA as compared to TCDD in the kidney and heart, and potentiated the TCDD-mediated induction of Gsta protein and activity in the kidney.

4.?In conclusion, TMAO modulates AhR-regulated genes in a tissue- and enzyme-specific manner.     

Fibroblast growth factor (Fgf) 21 is a novel target gene of the aryl hydrocarbon receptor (AhR)

The toxic effects of dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), mainly through activation of the aryl hydrocarbon receptor (AhR) are well documented. Fibroblast growth factor (Fgf) 21 plays critical roles in metabolic adaptation to fasting by increasing lipid oxidation and ketogenesis in the liver. The present study was performed to determine whether activation of the AhR induces Fgf21 expression. In mouse liver, TCDD increased Fgf21 mRNA in both dose- and time-dependent manners. In addition, TCDD markedly increased Fgf21 mRNA expression in cultured mouse and human hepatocytes. Moreover, TCDD increased mRNA (in liver) and protein levels (in both liver and serum) of Fgf21 in wild-type mice, but not in AhR-null mice. Chromatin immunoprecipitation assays showed that TCDD increased AhR protein binding to the Fgf21 promoter (− 105/+ 1 base pair). Fgf21-null mice administered 200 μg/kg of TCDD died within 20 days, whereas wild-type mice receiving the same treatment were still alive at one month after administration. This indicates that TCDD-induced Fgf21 expression protects against TCDD toxicity. Diethylhexylphthalate (DEHP) pretreatment attenuated TCDD-induced Fgf21 expression in mouse liver and white adipose tissue, which may explain a previous report that DEHP pretreatment decreases TCDD-induced wasting. In conclusion, Fgf21 appears to be a target gene of AhR-signaling pathway in mouse and human liver.     

Relationship between aryl hydrocarbon receptor-affinity and the induction of EROD activity by 2,3,7,8-tetrachlorinated phenothiazine and derivatives

Reported herein are semi-empirical calculations of the molecular geometry of TCDD, TCPT, TCPT-sulfoxide (TCPT-O), TCPT-sulfone (TCPT-O(2)), N-methyl-TCPT (Me-TCPT), N-methyl-TCPT-sulfoxide (Me-TCPT-O), and N-methyl-TCPT-sulfone (Me-TCPT-O(2)), the characterization of their AhR binding affinity in rat hepatic cytosol, and their ability to induce EROD activity in a rat hepatoma cell line in vitro. Semi-empirical calculations yielded detailed information about the stereochemistry and the preferred conformation of each of these compounds. These results in combination with observations reported in this paper were used to determine structure-activity relationships. In vitro displacement of (3)H-TCDD was measured by increasing concentrations of the respective ligands. This assay revealed a strong binding affinity of TCPT to the AhR with a K(i) value of 1.08 nM. TCDD had a K(i) value of 0.54 nM. The affinity of TCPT derivatives for the AhR decreased with increasing degree of oxidation. Moreover, N-methylation further lowered the affinity, so that the N-methyl sulfone derivative of TCPT displayed the highest K(i) at approximately 1200 nM (=460.4 ng/ml). A corresponding trend was observed regarding the potency of TCPT and derivatives to induce EROD activity in vitro. However, the potencies were considerably lower than that of TCDD. Enzyme induction was measured in a rat hepatoma cell line H4IIEC/T3 by quantification of ethoxyresorufin-O-deethylase (EROD) activity. Induction was measured at 12, 24, 48 and 72 h to determine time dependence. Sulfoxidated and N-methylated phenothiazines displayed a lower potency than their respective parent compounds. TCPT and all derivatives induced enzyme activity at an efficacy similar to TCDD at all time points measured. The reported findings clearly separate the induction of EROD activity by TCPT and derivatives from their binding affinities to the AhR. In contrast, a direct correlation between the two is generally assumed in drug development, leading to - in our view - unwarranted termination of drug candidates. Therefore, a lack of such a correlation for TCPT and derivatives in fact supports their further development as possible drug leads.     

Crosstalk between activated forms of the aryl hydrocarbon receptor and glucocorticoid receptor

Pyrene, benzo[a]pyrene (BaP), and indeno[1,2,3-cd]pyrene (IND) are poly cyclic aromatic hydrocarbons (PAHs) with four to six annealed phenyl rings. Dexamethasone (Dex) is a synthetic agonist of glucocorticoids. The aryl hydrocarbon receptor (AhR) ligands, BaP and IND, did not directly activate the glucocorticoid receptor (GR), and Dex did not activate the AhR either. Whenever BaP and IND were added to Dex-treated cultures, they were present with Dex for longer periods, and higher enhancement of Dex-induced transactivation of the GR was found, which indicates that the freshly activated AhR is essential for synergistic interactions with the activated GR. The degree of enhancement of Dex-induced transactivation of the GR by PAHs, BaP ≈ IND > pyrene, paralleled the potency of PAHs in activating the AhR. This synergistic interaction was more distinct in ovarian granulosa cells (HO23) than in HepG2, 293T, or HeLa cells. In contrast, Dex suppressed AhR-mediated expressions, including AhR and cytochrome P450 (CYP) 1 A1 expressions. Dex also counteracted the BaP-induced decrease in cell viability. Crosstalk between the AhR and GR was independent of their expression levels. We concluded that the AhR functionally cross-reacts with the GR, through which transactivation activity of the GR is further enhanced, and in contrast, transactivation activity of the AhR is inhibited. This report shows the significance of in vitro endocrine-related results, which provide a clue for molecular studies of an interactive mechanism between the AhR and GR, and should be confirmed by future in vivo studies.     

Preliminary screening of the inhibitory effect of food extracts on activation of the aryl hydrocarbon receptor induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin

A preliminary screening for the inhibitory effects on the activation of the aryl hydrocarbon receptor (AhR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by applying AhR-based bioassays for dioxins, the Ah-Immunoassay and CALUX assay, was attempted. Thirty-nine food extracts including vegetables, fruits, herbs, and teas were initially screened in vitro. We first examined the application of both bioassay methods using green tea extracts and (-)-epigallocatechin gallate, reported antagonists of the AhR, since the results could reveal an inhibitory effect versus the control in both assays. Food extracts were then tested. Among the herbs, extracts of sage, among the vegetables, green leafy ones such as spinach, and among the fruit, citrus showed inhibitory effects on AhR activation by TCDD, although some tested samples did not show parallel behavior in both assays. Sage had a remarkable inhibitory effect (79% in the CALUX assay and 83% in the Ah-Immunoassay compared with control) and its effects were dose dependent. The results suggest that these assays might be applicable to the preliminary screening of antagonist activity against the AhR. Moreover, based on these results, the potential benefit of factors that function as dietary ligands of the AhR and are present in several foodstuffs is indicated.