Expression Of Aromatic Hydrocarbon Receptor Research Articles

Elevated profiles of peripheral Th22, Th17, Th2 cells, and decreased percentage of Th1 cells in breast cancer patients.

Th22 subset is a particular type of CD4+ T helper cells subset. Our study aimed to explore the expression level of circulating Th22, Th17, Th1, and Th2 cells and the possible mechanism of these cells in breast cancer (BC) with different pathological features. Our study enrolled 43 newly diagnosed BC patients and 30 healthy controls. Frequencies of peripheral Th22, Th17, Th1, and Th2 cells were tested by flow cytometry. Concentrations of IL-22 cytokine in plasma were examined by enzyme-linked immunosorbent assay (ELISA). Real-time PCR was done to test aromatic hydrocarbon receptor (AHR) and RAR-associated orphan receptor C (RORC) gene expression. Frequencies of Th22, Th17, Th2 subsets, and the plasma IL-22 level was obviously higher in the BC patients. A positive correlation between Th22 frequency and IL-22 concentration in plasma was detected in BC patients. Furthermore, the percentage of Th22, Th2 subsets in peripheral blood of HER2 positive BC was higher than that in HER2 negative BC patients. A negative correlation between Th1 subset and Ki-67% as well as a positive correlation between Th2 subset and Ki-67% was found in BC patients. The proportion of Th1 cells in BC patients was significantly lower than that of the control group. Expression of AHR and RORC transcription factors were also observed to be upregulated in the BC patients. Furthermore, Th22 cells were positively correlated with BC tumor stage and clinical outcomes. The BC patients with a higher percentage of Th22, Th17, Th1 cells or a lower percentage of Th1 cells showed a decreased trend of survival rate. Th22, Th17, Th1, and Th2 subsets may play an essential role in BC patients. Th22, Th17, Th1, and Th2 cells may have potential significance to be used as clinical markers in BC patients with different molecular classification. Th22 cells may have potential value in BC patients' outcomes prediction, providing clinical value.

Prevention of polycystic ovary syndrome and postmenopausal osteoporosis by inhibiting apoptosis with Shenling Baizhu powder compound.

Shenling Baizhu powder (SBP) has been shown to reverse the abnormal expression of the aromatic hydrocarbon receptor (AHR) mediated by air pollution. Our study aimed to understand the main ingredient of SBP and investigate its action mechanism in preventing polycystic ovary syndrome (POCS) and postmenopausal osteoporosis (PMO). The active ingredients of SBP with the highest binding affinity to AHR were screened using a Chinese medicine database, and their binding mechanism was simulated using molecular dynamics simulation (MDS). Rutin was utilized to treat ovarian granulosa cell lines and osteoblast cell lines. The cell lines were treated with a gradient of rutin concentration (0.01 mmol/L, 0.05 mmol/L and 0.1 mmol/L) to find the optimal drug dose. PCR was used to detect AHR and apoptosis-related proteins, and WB to detect the expression of AHR, caspase-3 and cleaved-caspase-3. Finally, the CCK-8 cell proliferation assay detected the proliferation of cells. We obtained Rutin through the Chinese medicine database, and dynamics simulation determined its binding sites. Ovarian granulosa cell lines and osteoblast cell lines were treated with Rutin. RT-PCR and western blotting revealed that the expression of apoptosis-associated protein Bcl-2 was elevated, and the expression of AHR, Bax, caspase-3 and PARP were decreased. CCK-8 results showed accelerated proliferation in both cell types. Rutin, the main ingredient of SBP compound, works by binding to AHR, which can improve POCS and PMO by inhibiting cell apoptosis and by promoting cell proliferation.

An acetonic extract and secondary metabolites from the endolichenic fungus Nemania sp. EL006872 exhibit immune checkpoint inhibitory activity in lung cancer cell.

Background: Endolichenic fungi (ELF), which live the inside the lichen thallus, contain many secondary metabolites that show various biological activities. Recent studies show that lichen and ELF secondary metabolites have antioxidant, antibacterial, antifungal, cytotoxic, and anticancer activities. Purpose: Here, the effects of an ELF extract and its bioactive compounds were investigated on the H1975 cell line focusing on immune checkpoint marker inhibition. Methods: An ELF was isolated from the host lichen Bryoria fuscescens (Gyelnik) Brodo and D. Hawksw and identified the species as Nemania sp. EL006872. The fungus was cultured on agar medium and acetonic extracts were obtained. Secondary metabolites radianspenes C and D, and dahliane D, were isolated from the crude extract. The biological effects of both the crude extract and the isolated secondary metabolites were evaluated in cell viability, qRT-PCR assays, flow cytometry analysis and western blotting. Results: The cell viability assay revealed that extracts from Nemania sp. EL006872 and the isolated secondary compounds had low cytotoxicity. The crude extract, radianspenes C and D, and dahliane D, suppressed expression of mRNA encoding PD-L1 and aromatic hydrocarbon receptor (AhR), and surface expression of PD-L1 protein by cells exposed to benzo[a] pyrene. Radianspenes C and D, and dahliane D, reduced expression of AhR, PD-L1, ICOSL, and GITRL proteins by H1975 lung cancer cells, as well as exerting anti-proliferative effects. Conclusion: Radianspenes C and D, and dahliane D, bioactive compounds isolated from Nemania sp. EL006872 ELF, have the potential for use as immunotherapy and immunoncology treatments.

Cancer Associated Fibroblasts Promote Renal Cancer Progression Through a TDO/Kyn/AhR Dependent Signaling Pathway.

Cancer associated fibroblasts (CAFs) play crucial roles in cancer development, however, the specific mechanisms of CAFs associated renal cancer progression remain poorly understood. Our study observed enriched CAFs in high degree malignant tumor tissues from renal cancer patients. These CAFs isolated from tumor tissues are prone to facilitate drugs resistance and promote tumor progression in vitro and in vivo. Mechanistically, CAFs up-regulated tryptophan 2, 3-dioxygenase (TDO) expression, resulting in enhanced secretion of kynurenine (Kyn). Kyn produced from CAFs could up-regulated the expression of aromatic hydrocarbon receptor (AhR), eventually resulting in the AKT and STAT3 signaling pathways activation. Inhibition of AKT signal prevented cancer cells proliferation, while inhibition of the STAT3 signal reverted drugs resistance and cancer migration induced by kynurenine. Application of AhR inhibitor DMF could efficiently suppress distant metastasis of renal cancer cells, and improve anticancer effects of sorafenib (Sor)/sunitinib (Sun), which described a promising therapeutic strategy for clinical renal cancer.

Circulating Th22 cells, as well as Th17 cells, are elevated in patients with renal cell carcinoma

T-helper (Th) 22 cells serve an essential role in different types of tumors and autoimmune diseases. No research has been conducted to study the role of Th22 cells in the pathogenesis of renal cell carcinoma (RCC). We aimed to evaluate the prognostic value of circulating Th22, Th17, and Th1 cells in RCC patients. Thirty-two newly diagnosed RCC patients and thirty healthy controls were enlisted in the research. Their peripheral blood was collected, and the frequencies of circulating Th22, Th17, and Th1 cells were detected by flow cytometry. Plasma IL-22 concentrations were examined by an enzyme-linked immunosorbent assay (ELISA). Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to identify the mRNA expression levels of aromatic hydrocarbon receptor (AHR) and RAR-associated orphan receptor C (RORC) in peripheral blood mononuclear cells (PBMC). Compared with the healthy control group, the frequency of circulating Th22 and Th17 cells and concentrations of plasma IL-22 were significantly increased in RCC patients. However, there was no significant difference in the frequency of Th1 cells. A positive correlation between Th22 cells and plasma IL-22 levels was found in RCC patients. Also, there was a significant positive correlation between Th22 and Th17 cells in RCC patients. An up-regulated expression of AHR and RORC transcription factors were also observed in RCC patients. As tumor stage and grade progressed, the frequencies of Th22 and Th17 cells and the level of plasma IL-22 significantly increased. Meanwhile, there was a positive correlation between Th22 and Th17 cells and RCC tumor stage or grade. Furthermore, patients with high Th22 or Th17 cells frequency displayed a decreased trend in survival rate. Our research indicated that the increased circulating Th22 and Th17 cells and plasma IL-22 may be involved in the pathogenesis of RCC and may be involved in the occurrence and development of tumors. Th22 cells, plasma IL-22, and Th17 cells may be promising new clinical biomarkers and may be used as cellular targets for RCC therapeutic intervention.

Chlorpyrifos-induced cell proliferation in human breast cancer cell lines differentially mediated by estrogen and aryl hydrocarbon receptors and KIAA1363 enzyme after 24 h and 14 days exposure

Organophosphate biocide chlorpyrifos (CPF) is involved with breast cancer. However, the mechanisms remain unknown. CPF increases cell division in MCF-7 cells, by estrogen receptor alpha (ERα) activation, although it is a weak ERα agonist, suggesting other mechanisms should be involved. Aromatic hydrocarbon receptor (AhR) activation increases cell division in human breast cancer cells, and CPF strongly activates it. Finally, the KIAA1363 enzyme, which is regulated by CPF, is overexpressed in cancer cells. Accordingly, we hypothesized that CPF or its metabolite chlorpyrifos-oxon (CPFO) could induce cell viability promotion in MCF-7 and MDA-MB-231 cell lines, through mechanisms related to ERα, AhR, and KIAA1363, after 24 h and 14 days treatment. Results show that, after acute and long-term treatment, CPF and CPFO alter differently KIAA1363, AhR, ER and cytochrome P450 isoenzyme 1A1 (CYP1A1) expression. In addition, they induced cell proliferation through ERα activation after 24 h exposure in MCF-7 cells and through KIAA1363 overexpression and AhR activation in MCF-7 and MDA-MB-231 cells after acute and long-term treatment. The results obtained in this work provide new information relative to the mechanisms involved in the CPF toxic effects that could lead to breast cancer disease.

Constitutive expression of AhR and BRCA-1 promoter CpG hypermethylation as biomarkers of ERα-negative breast tumorigenesis.

BackgroundOnly 5–10 % of breast cancer cases is linked to germline mutations in the BRCA-1 gene and occurs early in life. Conversely, sporadic breast tumors, which represent 90-95 % of breast malignancies, have lower BRCA-1 expression, but not mutated BRCA-1 gene, and tend to occur later in life in combination with other genetic alterations and/or environmental exposures. The latter may include environmental and dietary factors that activate the aromatic hydrocarbon receptor (AhR). Therefore, understanding if changes in expression and/or activation of the AhR are associated with somatic inactivation of the BRCA-1 gene may provide clues for breast cancer therapy.MethodsWe evaluated Brca-1 CpG promoter methylation and expression in mammary tumors induced in Sprague–Dawley rats with the AhR agonist and mammary carcinogen 7,12-dimethyl-benzo(a)anthracene (DMBA). Also, we tested in human estrogen receptor (ER)α-negative sporadic UACC-3199 and ERα-positive MCF-7 breast cancer cells carrying respectively, hyper- and hypomethylated BRCA-1 gene, if the treatment with the AhR antagonist α-naphthoflavone (αNF) modulated BRCA-1 and ERα expression. Finally, we examined the association between expression of AhR and BRCA-1 promoter CpG methylation in human triple-negative (TNBC), luminal-A (LUM-A), LUM-B, and epidermal growth factor receptor-2 (HER-2)-positive breast tumor samples.ResultsMammary tumors induced with DMBA had reduced BRCA-1 and ERα expression; higher Brca-1 promoter CpG methylation; increased expression of Ahr and its downstream target Cyp1b1; and higher proliferation markers Ccnd1 (cyclin D1) and Cdk4. In human UACC-3199 cells, low BRCA-1 was paralleled by constitutive high AhR expression; the treatment with αNF rescued BRCA-1 and ERα, while enhancing preferential expression of CYP1A1 compared to CYP1B1. Conversely, in MCF-7 cells, αNF antagonized estradiol-dependent activation of BRCA-1 without effects on expression of ERα. TNBC exhibited increased basal AhR and BRCA-1 promoter CpG methylation compared to LUM-A, LUM-B, and HER-2-positive breast tumors.ConclusionsConstitutive AhR expression coupled to BRCA-1 promoter CpG hypermethylation may be predictive markers of ERα-negative breast tumor development. Regimens based on selected AhR modulators (SAhRMs) may be useful for therapy against ERα-negative tumors, and possibly, TNBC with increased AhR and hypermethylated BRCA-1 gene.

BRCA-1 promoter hypermethylation and silencing induced by the aromatic hydrocarbon receptor-ligand TCDD are prevented by resveratrol in MCF-7 Cells

Epigenetic mechanisms may contribute to reduced expression of the tumor suppressor gene BRCA-1 in sporadic breast cancers. Through environmental exposure and diet, humans are exposed to xenobiotics and food compounds that bind the aromatic hydrocarbon receptor (AhR). AhR-ligands include the dioxin-like and tumor promoter 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). The activated AhR regulates transcription through binding to xenobiotic response elements (XREs=GCGTG) and interactions with transcription cofactors. Previously, we reported on the presence of several XREs in the proximal BRCA-1 promoter and that the expression of endogenous AhR was required for silencing of BRCA-1 expression by TCDD. Here, we document that in estrogen receptor-α-positive and BRCA-1 wild-type MCF-7 breast cancer cells, the treatment with TCDD attenuated 17β-estradiol-dependent stimulation of BRCA-1 protein and induced hypermethylation of a CpG island spanning the BRCA-1 transcriptional start site of exon-1a. Additionally, we found that TCDD enhanced the association of the AhR; DNA methyl transferase (DNMT)1, DNMT3a and DNMT3b; methyl binding protein (MBD)2; and trimethylated H3K9 (H3K9me3) with the BRCA-1 promoter. Conversely, the phytoalexin resveratrol, selected as a prototype dietary AhR antagonist, antagonized at physiologically relevant doses (1 μmol/L) the TCDD-induced repression of BRCA-1 protein, BRCA-1 promoter methylation and the recruitment of the AhR, MBD2, H3K9me3 and DNMTs (1, 3a and 3b). Taken together, these observations provide mechanistic evidence for AhR agonists in the establishment of BRCA-1 promoter hypermethylation and the basis for the development of prevention strategies based on AhR antagonists.

Aromatic Hydrocarbon Receptor Down‐regulates MYCN Expression and Promotes Neural Differentiation of Neuroblastoma

Neuroblastoma (NB) is a heterogeneous neoplasm derived from sympathoadreanal lineage of the neural crest progenitor cells, and is the most common malignant disease of infancy. MYCN amplification is a prognostic factor of NB. It indicates a highly malignant disease and poor patient prognosis. However, how MYCN affects the NB cell behavior remains unclear. By DNA arrays and pathway analysis, aromatic hydrocarbon receptor (AHR) was found to reversely correlate with the MYCN expression in NB tumors. In addition, positive AHR expression by immunostaining of NB tumors predicted a favorable prognosis. AHR was known as the mediator of the toxicity of dioxin‐like compounds. However, recent studies demonstrated that AHR is important in regulating cell death, proliferation, and differentiation. We hypothesize that AHR could regulate MYCN expression in NB. Over‐expressed AHR promotes SK‐N‐DZ cell neuronal differentiation and down‐regulate the mRNA expression of MYCN. Our results suggested that AHR not only relates to the MYCN expression but also plays an important role in regulating tumorigenesis of NB.

The Effect of Aromatic Hydrocarbon Receptor on the Phenotype of the Hepa 1c1c7 Murine Hepatoma Cells in the Absence of Dioxin

The aromatic hydrocarbon receptor (AhR) mediates biological responses to certain exogenous ligands, such as the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and has also been demonstrated to modulate the cell cycle and differentiated state of several cell lines independently of exogenous ligands. In this study, we used DNA micorarray analysis to elucidate the profile of genes responsive to the expression of unliganded AhR by re-introducing AhR into an AhR-deficient mouse derivative (c19) of the mouse hepatoma cell line Hepa1c1c7. 22 gene products were up-regulated and 8 were down-regulated two-fold or more in c19 cells infected with a retroviral vector expressing mouse AhR. Surprisingly, expression of genes involved in cell proliferation or differentiation were not affected by introduction of AhR. AhR also did not restore expression of the albumin gene in c19 cells. Introduction of AhR into c12, a similar AhR-defective mouse hepatoma cell line, also did not restore albumin expression, and furthermore, did not lead to changes in cellular morphology or cell cycle parameters. These observations fail to support the notion that unliganded AhR regulates proliferation and differentiation of liver-derived cells.

Influence of smoke tar on mRNA expression of aromatic hydrocarbon receptor and cytochrome P4501A1 gene of mice lungs

To investigate the influence of the smoke tar on the expression of aromatic hydrocarbon receptor (AHR) and the cytochrome P4501Al (CYP1A1) gene of mice lungs. The smoke tar of 5.29, 10.58 and 15.87 mg/kg was administered intraperitoneally in mice respectively. RNA of mice lungs was got with RNA kit. RT-PCR technique was used for determining AHR and CYP1A1 gene expression with beta-actin as control. The AHR gene expression level was (0.554 +/- 0.023) for the mice intraperitoneally administered with 5.29 mg/kg smoke tar for 72 hours with the significant difference in gene expression level compared with the Tween-80 group (0.484 +/- 0.045) (P < 0.05). The AHR gene expression levels were (0.555 +/- 0.014), (0.606 +/- 0.051), and (0.566 +/- 0.014), (0.684 +/- 0.069) for the mice intraperitoneally administered with 10.58 and 15.87 mg/kg smoke tar for 48 hours and 72 hours respectively with the significant difference in gene expression level compared with the Tween-80 group (0.486 +/- 0.060, 0.484 +/- 0.045) (P < 0.05, P < 0.01). The CYP1Al gene expression levels were (1.535 +/- 0.021), (1.643 +/- 0.046) and (1.624 +/- 0.056), (1.739 +/- 0.038) respectively with the significant difference compared with the Tween-80 group (l.436 +/- 0.016, 1.404 +/- 0.036) (P < 0.01). The smoke tar can regulate up the expression of AHR and CYP1A1 gene at a certain dosage and time. The regulation of the smoke tar for the expression of AHR was earlier than for that of CYP1A1.

Aromatic Hydrocarbon Receptor Expression and Function in Liver of Hypophysectomized Male Rats

The aromatic hydrocarbon receptor (AHR) acts as a ligand-activated transcription factor that mediates many of the biological responses to aromatic hydrocarbons, such as 3-methylcholanthrene (MC) and 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD). Some toxic effects are thought to be the result of AHR-mediated changes in the expression of endocrine-related genes, such as the estrogen receptor and genes involved in cell growth and differentiation. Since little is known about endocrine factors that regulate AHR expression and function, we evaluated the effect of hypophysectomy (hypx) on these parameters in the liver of male rats. Cytosolic AHR immunoreactive protein was reduced in hypx rats to 61% of levels in sham-operated rats. Sucrose density gradient analysis of cytosolic AHR radioligand binding using a saturating concentration of [ 3H]TCDD showed that ligand binding was reduced in hypx rats to 31% of sham levels. TCDD showed similar potency for transforming cytosolic AHR from hypx and sham rats to a DNA-binding form; however, maximal AHR DNA-binding was reduced in hypx rats to 57% of sham levels, although this did not achieve statistical significance. These changes observed at the protein level were not accompanied by a corresponding decrease in hypx rats of mRNA for AHR or the AHR nuclear translocator. Despite this change in AHR protein level, hypx rats were not compromised in hepatic cytochrome P450 1A1 induction following in vivo administration of MC. These data indicate that the AHR signaling pathway is intact in hypx rats and that a pituitary factor regulates hepatic AHR expression at the protein level.

Estrous cycle-regulated expression of CYP1B1 mRNA in the rat ovary

CYP1B1, a member of the cytochrome P450 superfamily, is expressed constitutively in the steroidogenic tissues of mammals and is inducible by peptide hormones, cAMP and aromatic hydrocarbon receptor (AHR) ligands. The mechanism of induction of this cytochrome P450 is similar to that for CYP1A1, i.e. through the aromatic hydrocarbon receptor (AHR) signaling pathway. We have recently reported that CYP1B1, but not CYP1A1, is expressed in rat granulosa cells (GC) in the absence of any external stimulus. The induction of CYP1B1 mRNA in rat GC by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in vitro was followed by an increase in AHR and estrogen receptor (ER-β) RNA levels. Estrous cycle-dependent expression of AHR, AHR-nuclear translocator (ARNT) and ER-mRNAs in the rat ovary was reported. We suggest that CYP1B1 may play a major role in the regulation of rat ovarian function/cycle but until now this has been unexplored experimentally. The present study was therefore aimed at examining the expression of CYP1A1, CYP1B1 and ER-mRNA in rat ovarian tissues throughout the estrous cycle to establish any correlation in the expressions of these mRNAs in rat ovary. Total RNA was extracted from the ovary and liver of cycling adult rats and the mRNAs were analyzed using relative RT-PCR with gene-specific primers for the target mRNA and for RPL 19 or S16 primers as an internal control. The results indicated that in the ovary, CYP1B1 mRNA increased significantly on the evening of proestrus and dramatically decreased on the morning of estrus, while ER-mRNA remained unaltered throughout the estrous cycle. CYP1A1 mRNA in the ovary and both CYP1A1 and CYP1B1 mRNAs in the liver were undetectable. That the sudden decrease of ovarian CYP1B1 mRNA on the morning of estrus was not an effect of the LH surge was verified in vitro using our short-term GC culture model. GC prepared from rats super-stimulated with equine chorionic gonadotropin (eCG) were cultured for 6 h with or without LH and TCDD. It was observed that both CYP1A1 and CYP1B1 mRNAs were induced by TCDD with no apparent effect of LH. It is suggested that the high level of CYP1B1 mRNA expression on the evening of proestrus in rat ovary might be involved in metabolism of estrogens to catecholestrogen (a known effect of CYP1B1), and that expression is unaffected in GC by LH.

Quantification of Aromatic Hydrocarbon Receptor (AHR) and Related Genes by Calibrated Reverse Transcription-PCR in Blood Mononuclear Cells

The aromatic hydrocarbon ( Ah ) gene battery (1) comprises Ah receptor ( AHR ), its nuclear translocator ( ARNT ), and the genes that encode the detoxifying enzymes of phase I (e.g., cytochrome P4501A1, -1A2, and -1B1) and phase II. These enzymes and growth regulatory factors such as interleukin 1-β (IL-1β) are coordinately induced by polychlorinated dibenzo- p -dioxins and polycyclic aromatic hydrocarbons, toxic chemicals that are ubiquitous in ambient air and in cigarette smoke, via AHR (1)(2)(3)(4). Quantification of the regulation of AHR and related genes could be useful to monitor the degree of polycyclic aromatic hydrocarbon exposure and to understand the pathways to chemical carcinogenesis and atherosclerosis (5). The expression of AHR and related genes has been studied using Northern blot analysis (6) or quantitative reverse transcription-PCR (RT-PCR) using labeled primers (7), which are low-sensitivity techniques; competitive RT-PCR (8), which is more sensitive but quite laborious; and real-time PCR (9), which is expensive. An interesting technique, proposed for the first time by Tsai and Wiltbank (10) and considered later by Bor et al. (11), is called “calibrated ” RT-PCR; it is a competitive RT-PCR based on the addition of a fixed amount of internal standard (IS) to the sample and to a set of calibrators. The labor, time, and costs for this method are lower than those for classic competitive RT-PCR. We present calibrated RT-PCR assays to study the expression of AHR , ARNT , cytochrome P4501B1 ( CYP1B1 ), and IL-1β for the AHR gene battery and of β-actin ( β-ACT ) as housekeeping gene in peripheral blood mononuclear cells from 32 healthy subjects. Total RNA was extracted according to the method of Chomczynski and Sacchi (12) from 1 × 107 peripheral blood mononuclear cells in suspension isolated by step-gradient centrifugation on Histopaque. RNA was resuspended in 50 μL …

Estrous cycle-dependent changes in the expression of aromatic hydrocarbon receptor (AHR) and AHR-nuclear translocator (ARNT) mRNAs in the rat ovary and liver

The aromatic hydrocarbon receptor (AHR) and AHR nuclear translocator protein (ARNT) mediate the toxic effects of a wide variety of halogenated and polycyclic aromatic hydrocarbons. While it can be assumed that AHR has an endogenous function, its role in reproduction is currently undefined. The present study seeks to examine the regulation of AHR and ARNT mRNAs in liver and ovarian tissues across the rat estrous cycle. Message for hepatic AHR was increased significantly on the morning of proestrus, and decreased dramatically by the evening of proestrus; while hepatic ARNT mRNA was significantly decreased between diestrus and the morning of proestrus, and between the evening of proestrus and the morning of estrus. Ovarian AHR mRNA was unchanged from diestrus to proestrus, and was decreased on the evening of proestrus. Changes in the expression of ARNT mRNA mirrored changes in the liver. To assess interaction between the AHR- and estrogen-receptor (ER)-signaling pathways and to test the hypothesis that estrogen regulates AHR mRNA, 25-day-old female rats were injected with either 17β-estradiol, the ER antagonist ICI 182 780, or with vehicle, and hepatic AHR mRNA was measured. Treatment with estrogen or the estrogen antagonist did not alter the abundance of AHR mRNA in the liver. These data suggest that while estrogen may not be the key regulator of AHR mRNA expression, a factor associated with the rat reproductive cycle may be important in regulating the expression of both the AHR and ARNT genes in the ovary and liver.

Aromatic Hydrocarbon Receptor Interaction with the Retinoblastoma Protein Potentiates Repression of E2F-dependent Transcription and Cell Cycle Arrest

Polyhalogenated aromatic hydrocarbons, of which 2,3,7, 8-tetrachloro-p-dioxin (TCDD) is the prototype compound, elicit a variety of toxic, teratogenic, and carcinogenic responses in exposed animals and in humans. In cultured cells, TCDD shows marked effects on the regulation of cell cycle progression, including thymocyte apoptosis, induction of keratinocyte proliferation and terminal differentiation, and inhibition of estrogen-dependent proliferation in breast cancer cells. The presence of an LXCXE domain in the dioxin aromatic hydrocarbon receptor (AHR), suggested that the effects of TCDD on cell cycle regulation might be mediated by protein-protein interactions between AHR and the retinoblastoma protein (RB). Using the yeast two-hybrid system, AHR and RB were in fact shown to bind to each other. In vitro pull-down experiments with truncated AHR peptides indicated that at least two separate AHR domains form independent complexes with hypophosphorylated RB. Coimmunoprecipitation of whole cell lysates from human breast carcinoma MCF-7 cells, which express both proteins endogenously, revealed that AHR associates with RB in vivo only after receptor transformation and nuclear translocation. However, the AHR nuclear translocator and transcriptional heterodimerization partner, is not required for (nor is it a part of) the AHR.RB complexes detected in vitro. Ectopic expression of AHR and RB in human osteosarcoma SAOS-2 cells, which lack endogenous expression of both proteins, showed that AHR synergizes with RB to repress E2F-dependent transcription and to induce cell cycle arrest. Furthermore, AHR partly blocked T-antigen-mediated reversal of RB-dependent transcriptional repression. These results uncover a potential function for the AHR in cell cycle regulation and suggest that this function may be that of serving as an environmental sensor that signals cell cycle arrest when cells are exposed to certain environmental toxicants.

12-O-tetradecanoylphorbol-13-acetate upregulates the Ah receptor and differentially alters CYP1B1 and CYP1A1 expression in MCF-7 breast cancer cells

Elevated expression of cytochrome P450 1B1 (CYP1B1) and estradiol 4-hydroxylation have been reported to be biomarkers of tumorigenesis in humans. The aromatic hydrocarbon receptor (AhR) regulates expression of human cytochrome P450 1A1 (CYP1A1) and CYP1B1, 17beta-estradiol (E2) 2- and 4-hydroxylases, respectively. There is also evidence that expression of estrogen receptor alpha (ERalpha) potentiates CYP1A1 inducibility in breast cancer cells. To characterize these relationships further, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), which downregulates ERalpha, and the high-affinity AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the expression of AhR, ERalpha, CYP1A1, and CYP1B1 in MCF-7 human breast cancer cells. Treatment with TPA, which suppressed ERalpha mRNA levels, caused a greater than fourfold elevation of AhR mRNA and protein levels, whereas treatment with TCDD caused a decrease in AhR protein but no change in ERalpha or AhR mRNA levels. In MCF-7 cells treated with TPA prior to treatment with TCDD, the AhR mRNA level was elevated, the ERalpha mRNA level remained suppressed, and the ratio of CYP1B1 to CYP1A1 mRNA was increased compared with treatment with TCDD alone. A corresponding increase in the ratio of the rates of 4- to 2-hydroxylation pathways of E2 metabolism was also observed in response to pretreatment with TPA prior to the addition of TCDD. These results demonstrate differential regulation of the human CYP1A1 and CYP1B1 genes and provide a cellular model to investigate further the mechanisms that may be involved in the elevated expression of CYP1B1 in tumorigenesis.

Expression of Functional Aromatic Hydrocarbon Receptor and Aromatic Hydrocarbon Nuclear Translocator Proteins in Murine Bone Marrow Stromal Cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) acting through the aromatic hydrocarbon receptor (AhR) and its dimerization partner, the AhR nuclear translocator protein (arnt), elicits numerous toxicological effects including immunosuppression and thymic atrophy. Previous work has shown that TCDD alters bone marrow prothymocyte populations. These effects could be mediated at the lymphocyte level directly and/or through effects on bone marrow stromal cells, a population important in the support of lymphopoiesis. The purpose of this study was to characterize AhR and arnt expression in three murine bone marrow stromal cell lines (S17, M2-10B4, and BMS2) and in primary stromal cell cultures. Immunoblot analysis detected AhR protein in M2-10B4 and BMS2 cells. AhR protein was also detected in the primary cultures. Arnt protein could be detected in all cell cultures. Electrophoretic mobility shift assays detected TCDD-dependent dioxin-responsive element (DRE) binding in all three cell lines. DNA binding was sequence-specific and dependent on AhR, as demonstrated by the addition of unlabeled DRE DNA or of anti-AhR antibody. Results obtained with the primary cultures paralleled those seen with the stromal cell lines. The ED50for induction of TCDD-dependent DRE binding in M2-10B4 cells was 0.21 nM. TCDD treatment did not induce stromal P4501A1 mRNA expression but did increase P4501B1 mRNA levels in all three cell lines and in the primary cultures. These results indicate that murine bone marrow stromal cells express AhR and arnt proteins. Furthermore, these proteins are functional in terms of their DRE-binding ability and potential to regulate mRNA levels in a gene-specific fashion.

Constitutive activation of the aromatic hydrocarbon receptor.

The ligand-activated aromatic hydrocarbon receptor (AHR) dimerizes with the AHR nuclear translocator (ARNT) to form a functional complex that transactivates expression of the cytochrome P-450 CYP1A1 gene and other genes in the dioxin-inducible [Ah] gene battery. Previous work from this laboratory has shown that the activity of the CYP1A1 enzyme negatively regulates this process. To study the relationship between CYP1A1 activity and Ah receptor activation we used CYP1A1-deficient mouse hepatoma c37 cells and CYP1A1- and AHR-deficient African green monkey kidney CV-1 cells. Using gel mobility shift and luciferase reporter gene expression assays, we found that c37 cells that had not been exposed to exogenous Ah receptor ligands already contained transcriptionally active AHR-ARNT complexes, a finding that we also observed in wild-type Hepa-1 cells treated with Ellipticine, a CYP1A1 inhibitor. In CV-1 cells, transient expression of AHR and ARNT leads to high levels of AHR-ARNT-dependent luciferase gene expression even in the absence of an agonist. Using a green fluorescent protein-tagged AHR, we showed that elevated reporter gene expression correlates with constitutive nuclear localization of the AHR. Transcriptional activation of the luciferase reporter gene observed in CV-1 cells is significantly decreased by (i) expression of a functional CYP1A1 enzyme, (ii) competition with chimeric or truncated AHR proteins containing the AHR ligand-binding domain, and (iii) treatment with the AHR antagonist alpha-naphthoflavone. These results suggest that a CYP1A1 substrate, which accumulates in cells lacking CYP1A1 enzymatic activity, is an AHR ligand responsible for endogenous activation of the Ah receptor.

Regulation of the Aromatic Hydrocarbon Receptor (AHR) by In-Utero and Lactational Exposure to 2, 3, 7, 8-Tetrachlorodibenzo-p-Dioxin (TCDD).

The aromatic hydrocarbon receptor (AHR) mediates the toxicity of several halogenated aromatic hydrocarbons, including 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). In order to better understand the systemic endocrine disruption induced by AHR agonists, we examined the regulation of AHR by exposure to TCDD during gestation and lactation. Pregnant rats were given an oral dose of 1 μg TCDD/kg body weight or vehicle control on day 15 of gestation, and female pups sacrificed on post-natal day 21. Total RNA from ovaries, uteri, and hypothalami was immobilized on a nylon membrane and probed with a murine AHR cDNA. The gel mobility-shift assay was used to asses changes in the DNA-binding activity of AHR in vitro. Ovarian AHR mRNA and DNA-binding capability were decreased 2.1- and 1.7-fold, respectively; while in the uterus, there was a 1.3- and 1.5-fold increase in message and DNA binding. Hypothalamic mRNA for AHR showed no change, while there was a 1.7-fold increase in DNA-binding activity. Our results show that in-utero and lactational exposure to TCDD alters the expression of AHR in a tissue-specific fashion, and that this altered regulation may either be a part or a cause of the general endocrine disruption observed in these animals.