Involvement Of Aryl Hydrocarbon Receptor Research Articles

Targeting the aryl hydrocarbon receptor with FICZ regulates IL-2 and immune infiltration to alleviate Hashimoto's thyroiditis in mice

Hashimoto's thyroiditis (HT) is the most frequent autoimmune disorder. Growing work points to the involvement of aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor, in the regulation of immune homeostasis. However, the roles of AhR and its ligands in HT remains unclear. In this study, we leveraged public human database analyses to postulate that the AhR expression was predominantly in thyroid follicular cells, correlating significantly with the thyroid infiltration levels of multiple immune cells in HT patients. Using a thyroglobulin-induced HT mouse model and in vitro thyroid follicular epithelial cell cultures, we found a significant downregulation of AhR expression in thyrocytes both in vivo and in vitro. Conversely, activating AhR by FICZ, a natural AhR ligand, mitigated inflammation and apoptosis in thyrocytes in vitro and conferred protection against HT in mice. RNA sequencing (RNA-seq) of thyroid tissues indicated that AhR activation moderated HT-associated immune or inflammatory signatures. Further, immunoinfiltration analysis indicated that AhR activation regulated immune cell infiltration in the thyroid of HT mice, such as suppressing cytotoxic CD8+ T cell infiltration and promoting anti-inflammatory M2 macrophage polarization. Concomitantly, the expression levels of interleukin-2 (IL-2), a lymphokine that downregulates immune responses, were typically decreased in HT but restored upon AhR activation. In silico validation substantiated the binding interaction between AhR and IL-2. In conclusion, targeting the AhR with FICZ regulates IL-2 and immune infiltration to alleviate experimental HT, shedding new light on the therapeutic intervention of this prevalent disease.

Aryl Hydrocarbon Receptor Involvement in the Sodium-Dependent Glutamate/Aspartate Transporter Regulation in Cerebellar Bergmann Glia Cells.

Glutamate, the major excitatory neurotransmitter in the vertebrate brain, exerts its functions through the activation of specific plasma membrane receptors and transporters. Overstimulation of glutamate receptors results in neuronal cell death through a process known as excitotoxicity. A family of sodium-dependent glutamate plasma membrane transporters is responsible for the removal of glutamate from the synaptic cleft, preventing an excitotoxic insult. Glial glutamate transporters carry out more than 90% of the brain glutamate uptake activity and are responsible for glutamate recycling through the GABA/Glutamate/Glutamine shuttle. The aryl hydrocarbon receptor is a ligand-dependent transcription factor that integrates environmental clues through its ability to heterodimerize with different transcription factors. Taking into consideration the fundamental role of glial glutamate transporters in glutamatergic synapses and that these transporters are regulated at the transcriptional, translational, and localization levels in an activity-dependent fashion, in this contribution, we explored the involvement of the aryl hydrocarbon receptor, as a model of environmental integrator, in the regulation of the glial sodium-dependent glutamate/aspartate transporter. Using the model of chick cerebellar Bergmann glia cells, we report herein that the aryl hydrocarbon receptors exert a time-dependent decrease in the transporter mRNA levels and a diminution of its uptake activity. The nuclear factor kappa light chain enhancer of the activated B cell signaling pathway is involved in this regulation. Our results favor the notion of an environmentally dependent regulation of glutamate removal in glial cells and therefore strengthen the notion of the involvement of glial cells in xenobiotic neurotoxic effects.

Targeting of neuroblastoma cells through Kynurenine-AHR pathway inhibition.

Neuroblastoma poses significant challenges in clinical management. Despite its relatively low incidence, this malignancy contributes disproportionately to cancer-related childhood mortality. Tailoring treatments based on risk stratification, including MYCN oncogene amplification, remains crucial, yet high-risk cases often confront therapeutic resistance and relapse. Here, we explore the aryl hydrocarbon receptor (AHR), a versatile transcription factor implicated in diverse physiological functions such as xenobiotic response, immune modulation, and cell growth. Despite its varying roles in malignancies, AHR's involvement in neuroblastoma remains elusive. Our study investigates the interplay between AHR and its ligand kynurenine (Kyn) in neuroblastoma cells. Kyn is generated from tryptophan (Trp) by the activity of the enzymes indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2). We found that neuroblastoma cells displayed sensitivity to the TDO2 inhibitor 680C91, exposing potential vulnerabilities. Furthermore, combining TDO2 inhibition with retinoic acid or irinotecan (two chemotherapeutic agents used to treat neuroblastoma patients) revealed synergistic effects in select cell lines. Importantly, clinical correlation analysis using patient data established a link between elevated expression of Kyn-AHR pathway genes and adverse prognosis, particularly in older children. These findings underscore the significance of the Kyn-AHR pathway in neuroblastoma progression, emphasizing its potential role as a therapeutic target.

Proteomic insight towards key modulating proteins regulated by the aryl hydrocarbon receptor involved in ovarian carcinogenesis and chemoresistance

Gynecological malignancies pose a severe threat to female lives. Ovarian cancer (OC), the most lethal gynecological malignancy, is clinically presented with chemoresistance and a higher relapse rate. Several studies have highly correlated the incidence of OC to exposure to environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a process mainly mediated through activating the aryl hydrocarbon receptor (AhR). We have previously reported that exposure of OC cells to TCDD, an AhR activator, significantly modulated the expression of several genes that play roles in stemness and chemoresistance. However, the effect of AhR activation on the whole OC cell proteome aiming at identifying novel druggable targets for both prevention and treatment intervention purposes remains unrevealed. For this purpose, we conducted a comparative proteomic analysis of OC cells A2780 untreated/treated with TCDD for 24 h using a mass spectrometry-based label-free shotgun proteomics approach. The most significantly dysregulated proteins were validated by Western blot analysis. Our results showed that upon AhR activation by TCDD, out of 2598 proteins identified, 795 proteins were upregulated, and 611 were downregulated. STRING interaction analysis and KEGG-Reactome pathway analysis approaches identified several significantly dysregulated proteins that were categorized to be involved in chemoresistance, cancer progression, invasion and metastasis, apoptosis, survival, and prognosis in OC. Importantly, selected dysregulated genes identified by the proteomic study were validated at the protein expression levels by Western blot analysis. In conclusion, this study provides a better understanding of the the cross-talk between AhR and several other molecular signaling pathways and the role and involvement of AhR in ovarian carcinogenesis and chemoresistance. Moreover, the study suggests that AhR is a potential therapeutic target for OC prevention and maintenance. SignificanceTo our knowledge, this is the first study that investigates the role and involvement of AhR and its regulated genes in OC by performing a comparative proteomic analysis to identify the critical proteins with a modulated expression upon AhR activation. We found AhR activation to play a tumor-promoting and chemoresistance-inducing role in the pathogenesis of OC. The results of our study help to devise novel therapeutics for better management and prevention and open the doors to finding novel biomarkers for the early detection and prognosis of OC.

Transcriptomic analysis in zebrafish larvae identifies iron-dependent mitochondrial dysfunction as a possible key event of NAFLD progression induced by benzo[a]pyrene/ethanol co-exposure.

Non-alcoholic fatty liver disease (NAFLD) is a worldwide epidemic for which environmental contaminants are increasingly recognized as important etiological factors. Among them, the combination of benzo[a]pyrene (B[a]P), a potent environmental carcinogen, with ethanol, was shown to induce the transition of steatosis toward steatohepatitis. However, the underlying mechanisms involved remain to be deciphered. In this context, we used high-fat diet fed zebrafish model, in which we previously observed progression of steatosis to a steatohepatitis-like state following a 7-day-co-exposure to 43mM ethanol and 25nM B[a]P. Transcriptomic analysis highlighted the potent role of mitochondrial dysfunction, alterations in heme and iron homeostasis, involvement of aryl hydrocarbon receptor (AhR) signaling, and oxidative stress. Most of these mRNA dysregulations were validated by RT-qPCR. Moreover, similar changes were observed using a human in vitro hepatocyte model, HepaRG cells. The mitochondria structural and functional alterations were confirmed by transmission electronic microscopy and Seahorse technology, respectively. Involvement of AhR signaling was evidenced by using in vivo an AhR antagonist, CH223191, and in vitro in AhR-knock-out HepaRG cells. Furthermore, as co-exposure was found to increase the levels of both heme and hemin, we investigated if mitochondrial iron could induce oxidative stress. We found that mitochondrial labile iron content was raised in toxicant-exposed larvae. This increase was prevented by the iron chelator, deferoxamine, which also inhibited liver co-exposure toxicity. Overall, these results suggest that the increase in mitochondrial iron content induced by B[a]P/ethanol co-exposure causes mitochondrial dysfunction that contributes to the pathological progression of NAFLD.

Pharmacological inhibition of soluble epoxide hydrolase alleviates chronic neuropathic pain induced anhedonia symptoms: differential role of AhR and TSPO signaling

Patients who suffer from chronic pain have a higher incidence to develop depression, which needs effective treatments. TPPU, a soluble epoxide hydrolase (sEH) inhibitor, was reported to have analgesia and antidepressant effects in animal models, suggesting its potential improvement in this comorbidity. Here, we sought to investigate: (1) the occurrence of anhedonia symptoms in mice with chronic neuropathic pain and the sEH protein expression in different tissues, including central nervous system and peripheral tissues; (2) the effects of TPPU on nociceptive and anhedonia behaviors in anhedonia susceptible mice, and (3) the involvement of Aryl hydrocarbon receptor (AhR) signaling and translocator protein (TSPO) signaling as possible targets for this comorbidity. Spared nerve injury (SNI) was constructed to mimic chronic neuropathic pain (CNP) model, and that mice in SNI group were classified into chronic pain with or without anhedonia phenotype via hierarchical cluster analysis of sucrose preference test (SPT) results. Protein levels of sEH were increased in medial prefrontal cortex (mPFC), hippocampus, spinal cord, liver, kidney and gut, but not in anterior cingulate cortex (ACC), nucleus accumbens (NAc), striatum, cerebellum, heart, muscle and vessel in anhedonia susceptible mice as compared with sham or anhedonia resilient mice. Chronic treatment with TPPU (3 mg/kg, orally, once per day for 7 days) improved withdraw threshold and SPT scores in anhedonia susceptible mice. Interestingly, the antidepressant, but not analgesia effect, of TPPU seems to be pharmacologically abrogated by pretreatment with AhR agonist (FICZ, orally, 100 μg/kg) via regulating inflammatory cytokines and CYP1A1 levels in the serum as well as neuroinflammation in the microglia and astrocytes. Interestingly, the classical TSPO pathway, a crucial factor in the analgesia and antidepressant effects of TPPU, failed to show any difference in subjects who were treated with drugs targeted at the AhR. Collectively, our findings indicate that TPPU may alleviate depression‐like symptoms in CNP mice through two independent pathways: (1) the direct antidepressant effects via regulating neuroinflammation mediated by AhR, and (2) the improvement of allodynia and depressive symptoms via the TSPO signaling and downstream neurosteroids. Thus, AhR provides a potential therapeutic target for treatment of depression induced by chronic pain, and strategies targeting TSPO signaling could be a vital approach to alleviate the comorbidity of chronic pain and depression.Support or Funding InformationThis work was supported , in part, by grants from the National Natural Science Foundation of China (to A.L., 81771159 and 81571047; to C.Y., 81703482)The overview of our manuscript.Figure 1

Involvement of aryl hydrocarbon receptor in the cytotoxicity of corannulene and its derivatives

Despite numerous studies on the toxicities of planar polycyclic aromatic hydrocarbons (PAHs), very little is known about the toxicological profiles of non-planar PAHs. In the present study, the cytotoxicity of corannulene (COR), a typical bowl-shaped PAH with a myriad of applications in the area of material chemistry, and benzo[a]pyrene (BaP), a typical planar PAH with similar molecular weight, were systematically compared in various cell lines. Compared with BaP, exposure to COR resulted in less cytotoxic responses in both human (HepG2) and murine (Hepa1-6) hepatoma cells, which was characterized with a slower cellular accumulation as well as a weaker induction of cytochrome P450 1 (CYP1/Cyp1) isozymes. Knockdown of aryl hydrocarbon receptor (AhR) by siRNA attenuated the inductive effect of COR on CYP1A/Cyp1a mRNA levels in these two cell lines. Further analysis revealed that derivatization greatly influenced the cytotoxicity of COR, which was positively correlated with their binding affinities to the AhR, as demonstrated by in silico molecular docking. Overall, these results suggest that AhR appears to be involved in the cytotoxic responses of COR and its derivatives, providing a fundamental understanding of the biological effects of bowl-like PAHs.

Elastin-derived peptide VGVAPG affects the proliferation of mouse cortical astrocytes with the involvement of aryl hydrocarbon receptor (Ahr), peroxisome proliferator-activated receptor gamma (Pparγ), and elastin-binding protein (EBP)

During aging and ischemic and hemorrhagic stroke, elastin molecules are degraded and elastin-derived peptides are released into the brain microenvironment. Val-Gly-Val-Ala-Pro-Gly (VGVAPG) is a repeating hexapeptide in the elastin molecule. It is well documented that the peptide sequence binds with high affinity to elastin-binding protein (EBP) located on the cell surface, thereby transducing a molecular signal into the cell. The aim of our study was to investigate whether EBP, aryl hydrocarbon receptor (Ahr), and peroxisome proliferator-activated receptor gamma (Pparγ) are involved in VGVAPG-stimulated proliferation. Primary astrocytes were maintained in DMEM/F12 medium without phenol red, supplemented with 10 or 1% charcoal/dextran-treated fetal bovine serum (FBS). The cells were exposed to increasing concentrations of VGVAPG peptide, and resazurin reduction was measured. In addition, Glb1, Pparγ, and Ahr genes were silenced. After 48 h of exposure to 10 nM and 1 µM of VGVAPG peptide, the level of estradiol (E2) and the expression of Ki67 and S100B proteins were measured. The results showed that at a wide range of concentrations, VGVAPG peptide increased the metabolism of astrocytes depending on the concentration of FBS. After silencing of Glb1, Pparγ, and Ahr genes, VGVAPG peptide did not affect the cell metabolism which suggests the involvement of all the mentioned receptors in its mechanism of action. Interestingly, in the low-FBS medium, the silencing of Glb1 gene did not result in complete inhibition of VGVAPG-stimulated proliferation. On the other hand, in the medium with 10% FBS VGVAPG increased Ki67 expression after Pparγ silencing, whereas in the medium with 1% FBS VGVAPG decreased Ki67 expression. Following the application of Ahr siRNA, VGVAPG peptide decreased the production of E2 and increased the expression of Ki67 and S100B proteins.

Involvement of Aryl Hydrocarbon Receptor in L-Kynurenine-Mediated Parathyroid Hormone-Related Peptide Expression.

Parathyroid hormone-related peptide (PTHrP), produced by specific cancers such as lung cancer, profoundly influences the formation of bone metastatic lesions via the "vicious cycle" of tumor growth and bone resorption. The changes in gene expression regulated by the abnormal microenvironment components play key roles in maintaining the biological characteristics of cells, such as the organotropism of cancer metastasis. A recent study has shown that L-kynurenine (L-Kyn), one of microenvironment components, induced a substantial increase in the metastasis of lung cancer cells. What remains unclear, however, is the linkage between L-Kyn and bone metastatic lesions. In the present paper, we found that a significant upregulation of PTHrP expression was detected when 95D cells, a lung cancer cell line, were incubated with 50μM of L-Kyn. Meanwhile, L-Kyn (50/100μM) strongly strengthened aryl hydrocarbon receptor (Ahr) expression. Additionally, L-Kyn (50μM) increased the expression of the nuclear translocation of Ahr and cytochrome P450 1A1. Most importantly, the L-Kyn-induced upregulation of migration was significantly reduced when cells were co-incubated with siRNAAhr. Notably, the L-Kyn-mediated increase in PTHrP was also substantially attenuated upon siRNAAhr treatment in 95D cells. These results suggest that Ahr is involved in the L-Kyn-induced enhancement of PTHrP expression.

The anti-inflammatory effect of LMWF5A and N-acetyl kynurenine on macrophages: Involvement of aryl hydrocarbon receptor in mechanism of action

After a traumatic insult, macrophages can become activated leading to general inflammation at the site of injury. Activated macrophages are partially regulated by the aryl hydrocarbon receptor (AhR) which when activated suppresses inflammation by limiting the secretion of pro-inflammatory cytokines and promoting the over expression of immuno-modulatory mediators. This study aims to determine whether the low molecular weight fraction of 5% human serum albumin (LMWF5A) and N-acetyl kynurenine (NAK), an N-acetyl tryptophan (NAT) breakdown product in LMWF5A, can regulate inflammation by inhibiting macrophage activation through the AhR since kynurenine is a known AhR agonist. Using LCMS, we demonstrate that NAT is non-enzymatically degraded during accelerated aging of LMWF5A with high heat accelerating degradation. More importantly, NAK is a major degradation product found in LMWF5A. THP-1 monocytes were differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA) and pre-treated with 2-fold dilutions of LMWF5A or synthetic NAK with or without an AhR antagonist (CH223191) prior to overnight stimulation with lipopolysaccharide (LPS). Treatment with LMWF5A caused a 50–70% decrease in IL-6 release throughout the dilution series. A dose-response inhibition of IL-6 release was observed for NAK with maximal inhibition (50%) seen at the highest NAK concentration. Finally, an AhR antagonist partially blocked the anti-inflammatory effect of LMWF5A while completely blocking the effect of NAK. A similar inhibitory effect was observed for CXCL-10, but the AhR antagonist was not effective suggesting additional mechanisms for CXCL-10 release. These preliminary findings suggest that LMWF5A and NAK partially promote the suppression of activated macrophages via the AhR receptor. Therefore, LMWF5A, which contains NAK, is potentially a useful therapeutic in medical conditions where inflammation is prevalent such as trauma, sepsis, and wound healing.

Involvement of Aryl hydrocarbon receptor in myelination and in human nerve sheath tumorigenesis

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in xenobiotic metabolism. Plexiform neurofibromas (PNFs) can transform into malignant peripheral nerve sheath tumors (MPNSTs) that are resistant to existing therapies. These tumors are primarily composed of Schwann cells. In addition to neurofibromatosis type 1 (NF1) gene inactivation, further genetic lesions are required for malignant transformation. We have quantified the mRNA expression levels of AHR and its associated genes in 38 human samples. We report that AHR and the biosynthetic enzymes of its endogenous ligand are overexpressed in human biopsies of PNFs and MPNSTs. We also detect a strong nuclear AHR staining in MPNSTs. The inhibition of AHR by siRNA or antagonists, CH-223191 and trimethoxyflavone, induces apoptosis in human MPNST cells. Since AHR dysregulation is observed in these tumors, we investigate AHR involvement in Schwann cell physiology. Hence, we studied the role of AHR in myelin structure and myelin gene regulation in Ahr-/- mice during myelin development. AHR ablation leads to locomotion defects and provokes thinner myelin sheaths around the axons. We observe a dysregulation of myelin gene expression and myelin developmental markers in Ahr-/- mice. Interestingly, AHR does not directly bind to myelin gene promoters. The inhibition of AHR in vitro and in vivo increased β-catenin levels and stimulated the binding of β-catenin on myelin gene promoters. Taken together, our findings reveal an endogenous role of AHR in peripheral myelination and in peripheral nerve sheath tumors. Finally, we suggest a potential therapeutic approach by targeting AHR in nerve tumors.

Estrogen receptor-α and aryl hydrocarbon receptor involvement in the actions of botanical estrogens in target cells

Botanical estrogen (BE) dietary supplements are consumed by women as substitutes for loss of endogenous estrogens at menopause. To examine the roles of estrogen receptor α (ERα) and aryl hydrocarbon receptor (AhR) and their crosstalk in the actions of BEs, we studied gene regulation and proliferation responses to four widely used BEs, genistein, daidzein, and S-equol from soy, and liquiritigen from licorice root in breast cancer and liver cells. BEs and estradiol (E2), acting through ERα, stimulated proliferation, ERα chromatin binding and target-gene expression. BEs but not E2, acting through AhR, bound to xenobiotic response element-containing chromatin sites and enhanced AhR target-gene expression (CYP1A1, CYP1B1). While E2 and TCDD acted quite selectively through their respective receptors, BEs acted via both receptors, with their AhR activity moderated by negative crosstalk through ERα. Both ERα and AhR should be considered as mediators of the biology and pharmacology of BEs.

Mixed-ligand copper(II) complexes activate aryl hydrocarbon receptor AhR and induce CYP1A genes expression in human hepatocytes and human cell lines.

The effects of four copper(II) mixed-ligand complexes [Cu(qui1)(L)]NO3·H2O (1-3) and [Cu(qui2)(phen)]NO3 (4), where qui1=2-phenyl-3-hydroxy-4(1H)-quinolinone, Hqui2=2-(4-amino-3,5-dichlorophenyl)-N-propyl-3-hydroxy-4(1H)-quinolinone-7-carboxamide, L=1,10-phenanthroline (phen) (1), 5-methyl-1,10-phenanthroline (mphen) (2), bathophenanthroline (bphen) (3), on transcriptional activities of steroid receptors, nuclear receptors and xenoreceptors have been studied. The complexes (1-4) did not influence basal or ligand-inducible activities of glucocorticoid receptor, androgen receptor, thyroid receptor, pregnane X receptor and vitamin D receptor, as revealed by gene reporter assays. The complexes 1 and 2 dose-dependently induced luciferase activity in stable gene reporter AZ-AhR cell line, and this induction was reverted by resveratrol, indicating involvement of aryl hydrocarbon receptor (AhR) in the process. The complexes 1, 2 and 3 induced CYP1A1 mRNA in LS180 cells and CYP1A1/CYP1A2 in human hepatocytes through AhR. Electrophoretic mobility shift assay EMSA showed that the complexes 1 and 2 transformed AhR in its DNA-binding form. Collectively, we demonstrate that the complexes 1 and 2 activate AhR and induce AhR-dependent genes in human hepatocytes and cancer cell lines. In conclusion, the data presented here might be of toxicological importance, regarding the multiple roles of AhR in human physiology and pathophysiology.

Potential effects of polychlorinated biphenyls (PCBs) and selected organochlorine pesticides (OCPs) on immune cells and blood biochemistry measures: a cross-sectional assessment of the NHANES 2003-2004 data.

BackgroundPolychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) are widely distributed in the environment and may have adverse effects on the immune system.MethodsLipid adjusted serum levels of 19 Dioxin Like (DL), 17 Non Dioxin Like (NDL) PCBs, 5 OCPs, and measures of complete blood count and routine biochemistry profile were obtained from the NHANES 2003-2004 cycle. For each of the PCB/OCP variables, individuals were put into four exposure groups and blood markers were compared across these groups.ResultsSerum levels of PCBs and OCPs increased with age. Total white blood cell (WBC) count, red blood cells (RBC), hemoglobin, and hematocrit measures were lowest in the group with the highest serum PCBs. Results for the OCPs varied. For Mirex, WBC declined in the highest exposure; no significant differences were observed for p-p’-DDT or p-p’-DDE; and higher levels of WBC were observed at the highest exposure groups of serum trans-nonachlor and oxychlordane. Liver enzymes (AST, ALT, and GGT) were significantly higher in the highest exposure groups of PCBs/OCPs.ConclusionsWe observed significant associations between PCB/OCP levels and blood markers in the general population. All of the levels were within normal ranges but the consistency of results is remarkable and may reflect subclinical effects. Largest differences were observed for NDL PCBs. Thus, routine application of toxic equivalency factors, which assume dioxin like mechanisms and aryl hydrocarbon receptor involvement, may not adequately reflect the effects of NDL PCBs in the mixture.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-069X-13-114) contains supplementary material, which is available to authorized users.

Basal and benzo[a]pyrene-induced expression profile of phase I and II enzymes and ABC transporter mRNA in the early life stage of Chinese rare minnows (Gobiocypris rarus)

ATP-binding cassette (ABC) transporters together with phase I and II detoxification enzymes have been considered as included in a cellular detoxification system. Previous studies have highlighted the involvement of aryl hydrocarbon receptor (AHR) and Cyp1a in PAH-induced embryo toxicity. However, the response of other xenobiotic enzymes/transporters in PAH-mediated embryo toxicity is not fully characterized. In the present study, rare minnow embryos were exposed to 10 and 100µg/L BaP within 4h post-fertilization (hpf) up to 168 hpf. RNA was extracted at 24, 48, 96, and 168 hpf. The basal and BaP-induced expression of phase I enzyme genes (cyp1a, 1b1, and 1c1), phase II enzyme gene (gstm and ugt1a), and ABC transporter genes (abcb1, abcc1, abcc2, and abcg2) mRNA was determined using real-time PCR. Severe developmental defects (e.g., spinal deformities, pericardial and yolk-sac edema) were observed in the BaP treated groups. The basal expression showed that gstm was most strongly expressed, followed by abcb1, ugt1a, and abcc2, whereas cyp1a, 1b1, 1c1, and abcg2 showed weak expression. BaP significantly induced the mRNA expression of three CYP1s (cyp1a, 1b1, and 1c1) (p<0.05) and the ABC transporters (abcc1, abcc2, and abcg2) in a dose-dependent manner. However, the mRNA expression of Phase II enzymes (gstm, ugt1a) for the BaP treatments showed no significant difference with that of the controls. Furthermore, distinct induced patterns of these genes were observed during different exposure periods. Simultaneous up-regulation of the cyp and ABC transporter gene transcripts suggests that a possible involvement and cooperation in the detoxification process could provide protection against the BaP toxicity of rare minnows at the early life stage.

The Evolving Role of the Aryl Hydrocarbon Receptor (AHR) in the Normophysiology of Hematopoiesis

In addition to its role as a toxicological signal mediator, the Aryl Hydrocarbon Receptor (AHR) is also a transcription factor known to regulate cellular responses to oxidative stress and inflammation through transcriptional regulation of molecules involved in the signaling of nucear factor-erythroid 2-related factor-2 (Nrf2), p53 (TRP53), retinoblastoma (RB1), and NFκB. Recent research suggests that AHR activation of these signaling pathways may provide the molecular basis for understanding AHR's evolving role in endogenous developmental functions during hematopoietic stem-cell maintenance and differentiation. Recent developments into the hematopoietic roles for AHR are reviewed, aiming to reconcile divergent findings as to the endogenous function of AHR in hematopoiesis. Potential mechanistic explanations for AHR's involvement in hematopoietic differentiation are discussed, focusing on its known role as a cell cycle mediator and its interactions with Hypoxia-inducible transcription factor-1 alpha (HIF1-α). Understanding the physiological mechanisms of AHR activation and signaling have far reaching implications ranging from explaining the action of various toxicological agents to providing novel ways to expand stem cell populations ex vivo for use in transplant therapies.

Involvement of aryl hydrocarbon receptor in basal and 2,3,7,8-tetrachlorodibenzo- p-dioxin-induced expression of target genes in primary human hepatocytes

Aryl hydrocarbon receptor (AhR) is a drug-sensing receptor activated by environmental contaminants such as 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), and is known to drive regulation of target genes in various human cell types. Its involvement in TCDD-mediated regulation of target genes in human hepatocytes however remains to be formally demonstrated. To gain insights into this point, we have analyzed the effects of AhR silencing on the regulation of various genes targeted by TCDD in primary human hepatocytes and highly-differentiated human hepatoma HepaRG cells. Efficient AhR knocking-down was performed through dimethyl sulfoxide-based transfection of small-interfering RNAs targeting AhR (siAhR). SiAhR-transfected human hepatocytes or HepaRG cells, exposed to TCDD, were found to exhibit reduced mRNA expression of various TCDD-responsive genes, i.e. CYP1A1, CYP1A2, CYP1B1, ALDH3A1, IL17RB, FER1L3 and SLC7A5, when compared to TCDD-treated counterparts transfected with non-targeting small-interfering RNAs. AhR silencing was moreover shown to markedly counteract TCDD-mediated induction of CYP1A1/CYP1A2/CYP1B1-related ethoxyresorufin O-deethylase activity in both human hepatocytes and HepaRG cells. It also concomitantly decreased constitutive mRNA expression of some target genes such as CYP1A1, CYP1A2, CYP1B1 and ALDH3A1. Taken together, these data indicate that AhR plays a crucial role in both basal and TCDD-induced expression of target genes in human hepatocytes.

Dual suppression of adipogenesis by cigarette smoke through activation of the aryl hydrocarbon receptor and induction of endoplasmic reticulum stress

Cigarette smoking decreases body weight, whereas molecular mechanisms underlying this phenomenon have not been elucidated. In this report, we investigated regulation of adipogenesis by cigarette smoke and involvement of aryl hydrocarbon receptor (AhR) and endoplasmic reticulum (ER) stress. We found that cigarette smoke extract (CSE) inhibited differentiation of preadipocytes into adipocytes dose dependently. It was associated with a decrease in lipid accumulation, blunted expression of adipocyte markers (adiponectin, PPAR-gamma, and C/EBPalpha), and sustained expression of a preadipocyte marker MCP-1. CSE markedly induced activation of AhR, and AhR agonists (2,3,7,8-tetrachlorodibenzo-p-dioxin, benzo[a]pyrene and 3-methylcholanthrene) reproduced the inhibitory effect of CSE on adipocyte differentiation. Furthermore, knockout of the AhR gene or blockade of AhR by a dominant-negative mutant attenuated the suppressive effects of CSE on adipocyte differentiation. We also found that CSE induced ER stress in preadipocytes, and ER stress inducers (thapsigargin, tunicamycin, and A23187) reproduced the suppressive effect of CSE on the differentiation of preadipocytes. Interestingly, AhR agonists did not cause ER stress, and ER stress inducers did not activate AhR. These results suggested that cigarette smoke has the potential to inhibit adipocyte differentiation via dual, independent mechanisms, i.e., through activation of the AhR pathway and induction of the unfolded protein response.

Genistein inhibits glutamate-induced apoptotic processes in primary neuronal cell cultures: An involvement of aryl hydrocarbon receptor and estrogen receptor/glycogen synthase kinase-3β intracellular signaling pathway

Phytoestrogens prevent neuronal damage, however, mechanism of their neuroprotective action has not been fully elucidated. This study aimed to evaluate the effects of genistein on glutamate-induced apoptosis in mouse primary neuronal cell cultures. Glutamate (1 mM) enhanced caspase-3 activity and lactate dehydrogenase (LDH) release in the hippocampal, neocortical and cerebellar neurons in time-dependent manner, and these data were confirmed at the cellular level with Hoechst 33342 and calcein AM staining. Genistein (10–10,000 nM) significantly inhibited glutamate-induced apoptosis, and the effect of this isoflavone was most prominent in the hippocampal cells. Next, we studied an involvement of estrogen and aryl hydrocarbon receptors in anti-apoptotic effects of genistein. A high-affinity estrogen receptor antagonist, ICI 182, 780 (1 μM), reversed, whereas less specific antagonist/partial agonist, tamoxifen (1 μM), either intensified or partially inhibited genistein effects. Aryl hydrocarbon receptor antagonist, α-naphthoflavone (1 μM), exhibited a biphasic action: it enhanced genistein action toward a short-term exposure (3 h) to glutamate, but antagonized genistein action toward prolonged exposure (24 h) to that insult. SB 216763 (1 μM), which preferentially inhibits glycogen synthase kinase-3β (GSK-3β), potentiated genistein effects. These data point to strong effects of genistein at low micromolar concentrations in various brain tissues against glutamate-evoked apoptosis. Moreover, this study provided evidence for involvement of aryl hydrocarbon receptor and estrogen receptor/GSK-3β intracellular signaling pathway in anti-apoptotic action of genistein.

Possible involvement of aryl hydrocarbon receptor (AhR) in adult T-cell leukemia (ATL) leukemogenesis: constitutive activation of AhR in ATL

Human T-cell leukemia virus type 1 is the etiologic agent of adult T-cell leukemia (ATL), although the precise mechanism involved in the transformation process has not yet been defined. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that can influence cell proliferation and differentiation. We investigated the expression and activation of AhR in ATL. RT-PCR and Western blot analyses showed high expression levels of AhR in ATL cell lines. The elevated expression of AhR was in part attributable to the action of the viral transactivator protein, Tax. Interestingly, activation of the AhR was found in ATL cell lines in the absence of apparent exogenous ligands. Importantly, the increased expression and activation of AhR were also observed in some primary ATL cells. To our best knowledge, this is the first report to show the lymphoid malignancy having constitutive activation of AhR. A possible link between increased AhR expression and leukemogenesis in ATL is discussed.