Aryl Hydrocarbon Receptor Nuclear Translocator mRNA Research Articles

Levels of aryl hydrocarbon receptor nuclear translocator in lung tissue of children with congenital heart disease pulmonary hypertension and its effect on proliferation and migration of pulmonary artery smooth muscle cells

Objective To observe the levels of aryl hydrocarbon receptor nuclear translocator (ARNT) in lung tissue of children with congenital heart disease (CHD) pulmonary artery hypertension (PAH) and its effect on proliferation and migration of human pulmonary artery smooth muscle cells (HPASMCs), and to investigate the action mechanism of ARNT in the pathogenesis of CHD-PAH. Methods Sixty patients with CHD ventricular septal defect-associated PAH were selected as CHD-PAH group and 60 children with CHD ventricular septal defect without PAH were selected as CHD group (CHD group) in our hospital. There were no significant difference in age and gender between the two groups. HPASMCs were divided into normoxia group and hypoxia group, which were induced by normoxia and hypoxia respectively. HPASMCs were divided into blank control group (BC group), negative control group (NC group) and siR-ARNT group. The cells in the siR-ARNT group were transfected with ARNT-shRNA lentivirus, and those in the NC group were transfected with negative control lentivirus. The cells in the BC group were not transfected. The expression levels of ARNT protein and mRNA in lung tissue and cells were detected by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The cell proliferation ability was determined by the cell counting kit-8 (CCK-8) method. Transwell and scratch assays were used to measure cell migration ability. SPSS 20.0 software was used for analysis. The test methods were t test and analysis of variance. Results The expression levels of ARNT protein and mRNA in lung tissue of CHD-PAH group (0.53±0.06 and 2.36±0.17) were significantly higher than those in CHD group (0.09±0.02, 1.00±0.12, t=53.889 and 50.626, P 0.05). Conclusion The ARNT levels in lung tissue of children with CHD-PAH are elevated. ARNT may participate in the development of CHD-PAH by affecting the proliferation and migration of HPASMCs. Key words: Congenital heart disease; Pulmonary hypertension; Aryl hydrocarbon receptor nuclear translocation protein; Pulmonary artery smooth muscle cells; Proliferation; Migration

Myeloid cell deletion of Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) induces non-alcoholic steatohepatitis.

Background and aimNon-alcoholic steatohepatitis (NASH) is predicted to become the most common cause of cirrhosis and liver failure. Risk factors include obesity, insulin resistance and diabetes. Macrophages and other myeloid cells play crucial roles in initiating and driving inflammation. Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) is a transcription factor which binds to a range of partners to mediate responses to environmental signals, including the diet. In people with diabetes it is decreased in liver. We hypothesised that myeloid cell ARNT activity may contribute to the development of liver pathology.MethodsFloxed-ARNT mice were bred with LysM-Cre mice to generate mice with reduced ARNT in myeloid cells. Animals were fed a high fat diet (HFD) and liver pathology was assessed. Histology, mRNA, fat accumulation and metabolism were studied.ResultsAnimals with reduced myeloid ARNT developed steatohepatitis on a HFD, with additional alterations of metabolism and fat deposition. Steatohepatitis was accompanied by hepatic macrophage infiltration and expression of both M1 and M2 markers. Expression of mRNAs for Cxcl1, Mcp-1, Tnf-α and Tgf-β1 were increased. Human livers from controls and people with NASH were tested; ARNT mRNA was decreased by 80% (p = 0.0004).ConclusionsDecreased myeloid ARNT may play a role in the conversion from non-alcoholic fatty liver to steatohepatitis. Increasing ARNT may be a therapeutic strategy to reduce NASH.

Molecular cloning and characterization of two ARNT (ARNT‐1 and ARNT‐2) genes in Atlantic croaker and their expression during coexposure to hypoxia and PCB77

Aryl hydrocarbon receptor nuclear translocator (ARNT) is an important transcriptions factor that binds/coactivates drug-metabolizing genes in vertebrates. In this study, we report the cloning and characterization of two ARNT (ARNT-1 and ARNT-2) genes and their mRNA and protein expression in liver tissues of Atlantic croaker after co-exposure to hypoxia and 3,3',4,4'-tetrachlorobiphenyl (PCB77). The full-length croaker ARNT-1 and ARNT-2 genes encode proteins of 537 and 530 amino acids, respectively, and are highly homologous to ARNT-1 and ARNT-2 genes of other vertebrates. ARNT mRNAs are ubiquitously expressed in all tissues. Hypoxia (dissolved oxygen: 1.7 mg/L) exposure (1-4 weeks) did not affect hepatic ARNTs mRNA levels. Dietary PCB77 treatment (2 and 8 μg/g body weight/day for 4 weeks) caused marked increases in ARNTs mRNA and protein levels in normoxic fish. However, coexposure to hypoxia and PCB77 for 4 weeks significantly blunted the increase in ARNTs mRNA and protein levels in response to PCB77 exposure. These results suggest that ARNT activity and functions induced by exposure to PCB aryl hydrocarbon receptor (AhR) agonists could be compromised in croaker inhabiting hypoxic coastal regions.

Comparative liver accumulation of dioxin-like compounds in sheep and cattle: Possible role of AhR-mediated xenobiotic metabolizing enzymes

PCDDs, PCDFs, and PCBs are persistent organic pollutants (POPs) that accumulate in animal products and may pose serious health problems. Those able to bind the aryl hydrocarbon receptor (AhR), eliciting a plethora of toxic responses, are defined dioxin-like (DL) compounds, while the remainders are called non-DL (NDL). An EFSA opinion has highlighted the tendency of ovine liver to specifically accumulate DL-compounds to a greater extent than any other farmed ruminant species. To examine the possible role in such an accumulation of xenobiotic metabolizing enzymes (XME) involved in DL-compound biotransformation, liver samples were collected from ewes and cows reared in an area known for low dioxin contamination. A related paper reported that sheep livers had about 5-fold higher DL-compound concentrations than cattle livers, while the content of the six marker NDL-PCBs did not differ between species. Specimens from the same animals were subjected to gene expression analysis for AhR, AhR nuclear translocator (ARNT) and AhR-dependent oxidative and conjugative pathways; XME protein expression and activities were also investigated. Both AhR and ARNT mRNA levels were about 2-fold lower in ovine samples and the same occurred for CYP1A1 and CYP1A2, being approximately 3- and 9-fold less expressed in sheep compared to cattle, while CYP1B1 could be detectable in cattle only. The results of the immunoblotting and catalytic activity (most notably EROD) measurements of the CYP1A family enzymes were in line with the gene expression data. By contrast, phase II enzyme expression and activities in sheep were higher (UGT1A) or similar (GSTA1, NQO1) to those recorded in cattle. The overall low expression of CYP1 family enzymes in the sheep is in line with the observed liver accumulation of DL-compounds and is expected to affect the kinetics and the dynamics of other POPs such as many polycyclic aromatic hydrocarbons, as well as of toxins (e.g. aflatoxins) or drugs (e.g. benzimidazole anthelmintics) known to be metabolized by those enzymes.

Role of glucocorticoid receptor and pregnane X receptor in dexamethasone induction of rat hepatic aryl hydrocarbon receptor nuclear translocator and NADPH‐cytochrome P450 oxidoreductase (LB597)

The aryl hydrocarbon receptor (AHR) nuclear translocator (ARNT), as the AHR’s heterodimerization partner, and NADPH‐cytochrome P450 oxidoreductase (POR), as the key electron donor for all microsomal P450s, are important players in the adaptive and toxic responses to polycyclic and halogenated aromatic hydrocarbons. Expression of both ARNT and POR in rat liver is induced by dexamethasone (DEX), a synthetic glucocorticoid known to activate both the glucocorticoid receptor (GR) and the pregnane X receptor (PXR). To study the role of GR in ARNT and POR regulation, we treated male rats with a low DEX dose (0.5 mg/kg), shown to activate GR selectively, and a high DEX dose (50 mg/kg), shown to activate both GR and PXR, in the absence and presence of the GR antagonist RU486 (50 mg/kg). The role of PXR was assessed by comparing the responses to similar low (1 mg/kg) and high (50 mg/kg) DEX doses in wild‐type and PXR‐knockout rats. In both studies, rats were euthanized at 6 and 24 h after DEX dosing. The induction of ARNT mRNA levels by low‐dose DEX and the increase in ARNT protein levels by high‐dose DEX were prevented by RU486, whereas the induced levels of ARNT mRNA and protein did not differ between wild‐type and PXR‐knockout rats. RU486 did not inhibit the induction by DEX of POR expression at the mRNA, protein, and catalytic activity levels. The induction of POR mRNA levels by low‐ and high‐dose DEX was significantly attenuated in PXR‐knockout rats and the increase in POR protein levels and catalytic activity by high‐dose DEX was completely absent in PXR‐knockout rats. These results are consistent with important roles for GR in the DEX induction of rat hepatic ARNT and PXR in the DEX induction of rat hepatic POR.Grant Funding Source: Supported by the Canadian Institutes of Health Research

Induction of the rat hepatic aryl hydrocarbon receptor nuclear translocator by glucocorticoids

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a shared partner between the hypoxia signaling pathway and the dioxin‐responsive aryl hydrocarbon receptor signaling pathway. Dexamethasone (DEX), a synthetic glucocorticoid known to activate both the glucocorticoid receptor (GR) and the pregnane X receptor (PXR), was shown previously to increase rat hepatic ARNT mRNA levels. To examine the roles of GR and PXR in this response, we treated male rats with the GR‐selective agonist triamcinolone acetonide (TA), the PXR‐selective agonist pregnenolone‐16α‐carbonitrile (PCN), and several doses of DEX. At 6 h following TA treatment, there was a 7.5‐fold induction of ARNT mRNA and a 4.5‐fold increase in levels of an unidentified lower molecular weight protein reacting with ARNT antibody. There was a trend for increased ARNT protein levels at 24 h after TA exposure. PCN treatment had no effect on these ARNT parameters. A low dose of DEX (1 mg/kg), shown to activate GR but not PXR, caused a 10‐fold induction of ARNT mRNA at 6 h along with a 6.5‐fold increase in levels of the unidentified ARNT antibody‐reactive protein. A trend for increased ARNT protein levels at 24 h was observed, but only at higher doses of DEX (10 to 50 mg/kg), shown to activate both GR and PXR. These findings suggest that the GR is a key mediator of the induction of rat hepatic ARNT expression by glucocorticoids. [Support: CIHR]

Aryl hydrocarbon receptor nuclear translocator in human liver is regulated by miR-24

Aryl hydrocarbon receptor nuclear translocator (ARNT) forms a heterodimer with aryl hydrocarbon receptor or hypoxia inducible factor 1α to mediate biological responses to xenobiotic exposure and hypoxia. Although the regulation mechanism of the ARNT expression is largely unknown, earlier studies reported that the human ARNT protein level was decreased by hydrogen peroxide or reactive oxygen species. These stimuli increase the miR-24 level in various human cell lines. In silico analysis predicts that some microRNAs including miR-16 and miR-23b may bind to ARNT mRNA. This background prompted us to investigate whether human ARNT is regulated by microRNAs. Overexpression of miR-24 into HuH-7 and HepG2 cells significantly decreased the ARNT protein level, but not the ARNT mRNA level, indicating translational repression. However, overexpression of miR-16 or miR-23b caused no change in the ARNT expression. The miR-24-dependent down-regulation of ARNT decreased the expression of its downstream genes such as CYP1A1 and carbonic anhydrase IX. Luciferase assay was performed to determine the element on the ARNT mRNA to which miR-24 binds. Finally, it was demonstrated that the miR-24 levels in a panel of 26 human livers were inversely correlated with the protein levels or the translational efficiency of ARNT. Taken together, we found that miR-24 negatively regulates ARNT expression in human liver, affecting the expression of its downstream genes. miR-24 would be one of the factors underlying the mechanisms by which ARNT protein is decreased by reactive oxygen species.

RNA expressions of AHR, ARNT and CYP1B1 are influenced by AHR Arg554Lys polymorphism

AimThe aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that together with Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) controls the expression of Xenobiotic metabolising enzymes (XME) such as CYP1B1. In the absence of exogenous ligands, AHR is supposed to be involved in promotion of cell cycle progression.Polymorphisms of the AHR gene are suggested to be associated with susceptibility to cancer. Because of its critical role in xenobiotic induced toxicity and carcinogenesis as well as its ligand independent relevance we investigated the effects of AHR Arg554Lys Polymorphism on gene expression level of the AHR, ARNT and CYP1B1. MethodsDetection of the AHR Arg554Lys polymorphism of the AHR gene was performed by rapid capillary PCR with melting curve analysis. The quantitative Real-Time PCR (qRT-PCR) of AHR, ARNT and CYP1B1 mRNAs was carried out in white blood cells from 287 Caucasians. Calculations of expression were made with the 2−ΔΔCT method. ResultsThe relative AHR mRNA expression revealed significant differences between the two homozygote AHR genotypes Arg554Arg (11.0±1.0; n=228) and Lys554Lys (0.6±0.4; n=3; p<0.001). Also significant differences were seen between the heterozygote genotype Arg554Lys (13.0±3.0; n=40) and the homozygote Lys554Lys genotype (0.6±0.4; n=3; p<0.001). These differences above were replicated significantly in the relative mRNA expression of ARNT and CYP1B1. Comparing the determined CT-values, a correlation coefficient of R=0.748 for AHR and ARNT, R=0.626 for ARNT and CYP1B1 as well as R=0.533 for AHR and CYP1B1 was calculated. ConclusionOur findings suggest that the homozygote variant genotype of AHR Lys554Lys is associated with a significantly lower AHR, ARNT and CYP1B1 mRNA expression.

Carbohydrate-Responsive Element-Binding Protein (ChREBP) Is a Negative Regulator of ARNT/HIF-1β Gene Expression in Pancreatic Islet β-Cells

OBJECTIVECarbohydrate-responsive element-binding protein (ChREBP) is a transcription factor that has been shown to regulate carbohydrate metabolism in the liver and pancreatic β-cells in response to elevated glucose concentrations. Because few genes have been identified so far as bona fide ChREBP-target genes, we have performed a genome-wide analysis of the ChREBP transcriptome in pancreatic β-cells.RESEARCH DESIGN AND METHODSChromatin immunoprecipitation and high-density oligonucleotide tiling arrays (ChIP-chip; Agilent Technologies) using MIN6 pancreatic β-cell extracts were performed together with transcriptional and other analysis using standard techniques.RESULTSOne of the genes identified by ChIP-chip and linked to glucose sensing and insulin secretion was aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia-inducible factor-1β (HIF-1β), a transcription factor implicated in altered gene expression and pancreatic-islet dysfunction in type 2 diabetes. We first confirmed that elevated glucose concentrations decreased ARNT/HIF-1β levels in INS-1 (832/13) cells and primary mouse islets. Demonstrating a role for ChREBP in ARNT gene regulation, ChREBP silencing increased ARNT mRNA levels in INS-1 (832/13) cells, and ChREBP overexpression decreased ARNT mRNA in INS-1 (832/13) cells and primary mouse islets. We demonstrated that ChREBP and Max-like protein X (MLX) bind on the ARNT/HIF-1β promoter on the proximal region that also confers the negative glucose responsiveness.CONCLUSIONSThese results demonstrate that ChREBP acts as a novel repressor of the ARNT/HIF-1β gene and might contribute to β-cell dysfunction induced by glucotoxicity.

Glucocorticoid and adrenalectomy effects on the rat aryl hydrocarbon receptor pathway depend on the dosing regimen and post-surgical time

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the effects of aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo- p-dioxin and 3-methylcholanthrene (MC); the prototypical response is induction of drug-metabolizing enzymes. Factors that regulate AHR levels in vivo are poorly understood and it is also not clear how AHR levels affect aromatic hydrocarbon responsiveness. Our interest in pituitary-dependent regulation of AHR levels was prompted by two findings from our laboratory: (1) hypophysectomized rats have reduced hepatic levels of AHR protein; and (2) glucocorticoids increase AHR expression and aromatic hydrocarbon responsiveness in rodent hepatoma cells. To study whether adrenalectomy and glucocorticoids contribute to hormone-dependent regulation of the hepatic AHR pathway, male adrenalectomized (ADX) or SHAM-ADX rats were treated with dexamethasone (DEX) or vehicle. AHR protein was depleted by 50–60% at 4 days after ADX, but was not altered by DEX treatment. To assess whether the observed AHR depletion affected aromatic hydrocarbon responsiveness, the induction of hepatic cytochrome P450 1B1 (CYP1B1) mRNA by MC was measured as an AHR-mediated adaptive response. MC-induced hepatic CYP1B1 mRNA was reduced by 50% in ADX rats relative to SHAM-ADX. Exogenous glucocorticoid treatment (DEX – 1.5 mg/kg) induced hepatic AHR nuclear translocator (ARNT) mRNA by up to 9-fold at 3 and 6 h after dosing, with no corresponding change in ARNT protein levels. These data demonstrate that: (1) adrenal-dependent factors contribute to the physiological maintenance of hepatic AHR protein levels; (2) the depletion of hepatic AHR protein in ADX rats coincided with a diminished adaptive response to MC; and (3) exogenous glucocorticoid treatment increases hepatic ARNT mRNA levels regardless of adrenal status. This model is useful for studying the mechanisms of AHR and ARNT regulation and for further characterization of the impact of AHR protein depletion on the response to aromatic hydrocarbons in vivo.

Aryl hydrocarbon receptor nuclear translocator (ARNT) gene as a positional and functional candidate for type 2 diabetes and prediabetic intermediate traits: Mutation detection, case-control studies, and gene expression analysis.

BackgroundARNT, a member of the basic helix-loop-helix family of transcription factors, is located on human chromosome 1q21–q24, a region which showed well replicated linkage to type 2 diabetes. We hypothesized that common polymorphisms in the ARNT gene might increase the susceptibility to type 2 diabetes through impaired glucose-stimulated insulin secretion.MethodsWe selected 9 single nucleotide polymorphisms to tag common variation across the ARNT gene. Additionally we searched for novel variants in functional coding domains in European American and African American samples. Case-control studies were performed in 191 European American individuals with type 2 diabetes and 187 nondiabetic European American control individuals, and in 372 African American individuals with type 2 diabetes and 194 African American control individuals. Metabolic effects of ARNT variants were examined in 122 members of 26 European American families from Utah and in 225 unrelated individuals from Arkansas. Gene expression was tested in 8 sibling pairs discordant for type 2 diabetes.ResultsNo nonsynonymous variants or novel polymorphisms were identified. No SNP was associated with type 2 diabetes in either African Americans or European Americans, but among nondiabetic European American individuals, ARNT SNPs rs188970 and rs11204735 were associated with acute insulin response (AIRg; p =< 0.005). SNP rs2134688 interacted with body mass index to alter β-cell compensation to insulin resistance (disposition index; p = 0.004). No significant difference in ARNT mRNA levels was observed in transformed lymphocytes from sibling pairs discordant for type 2 diabetes.ConclusionCommon ARNT variants are unlikely to explain the linkage signal on chromosome 1q, but may alter insulin secretion in nondiabetic subjects. Our studies cannot exclude a role for rare variants or variants of small (< 1.6) effect size.

Aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT) expression in Baikal seal ( Pusa sibirica) and association with 2,3,7,8-TCDD toxic equivalents and CYP1 expression levels

Most toxic effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons (PHAHs) are mediated by ligand-activated aryl hydrocarbon receptor (AHR) signaling pathway. To understand the regulation mechanism of AHR and AHR nuclear translocator (ARNT) expression in wild Baikal seal ( Pusa sibirica) population contaminated by PHAHs, the present study investigated hepatic mRNA expression levels of AHR and its heterodimer, ARNT genes, in association with biological index (age, gender and body weight), PHAH accumulation and expression levels of cytochrome P450 (CYP) 1A and 1B. While there was no gender difference, the AHR mRNA expression levels were increased with ages ( p = 0.014) and body weights ( p = 0.015), indicating that AHR expression might be affected by these biological factors. The AHR mRNA expression levels exhibited significant positive correlations with total TEQs and most of individual congener TEQs derived from polychorinated dibenzo- p-dioxins, dibenzofurans and non- ortho coplanar polychorinated biphenyls (PCBs), indicating the transcriptional up-regulation of AHR expression by these congeners. On the other hand, there was no significant correlation between individual TEQs from mono -ortho coplanar PCBs and AHR expression. These results imply the structure-related transcriptional activity of AHR among PHAHs congeners. AHR mRNA levels showed positive correlations with both CYP1A protein ( p = 0.039) and CYP1A1 mRNA expression levels ( p = 0.046). In contrast to AHR expression, neither the total nor individual congener TEQs influenced ARNT at the transcriptional level. ARNT mRNA showed significant negative correlations with CYP1A/1B protein ( p = 0.027 and p = 0.006) and CYP1A1 mRNA expression levels ( p = 0.039), implying the existence of different transcriptional regulation between AHR and ARNT genes and negative regulation by CYP1A/1B-mediated signaling pathways. The present findings may render significant insight on the basic mechanisms underlying regulation of AHR and ARNT expressions associated with biological factors and PHAH exposure in wild mammalian populations.

Uterine and ovarian aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) mRNA expression in benign and malignant gynaecological conditions.

The transcriptional regulators aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) modulate the transcription of genes involved in cellular differentiation and proliferation. In this study, we investigated the expression of these transcriptional regulators in the female reproductive tract. AHR and ARNT mRNA transcripts were readily detected by a ribonuclease protection assay in all reproductive tissues examined. The expression of these factors in the endometrium and myometrium did not vary during the menstrual cycle, and was not different in pre- versus post-menopausal women. However, post-menopausal women on continuous hormone replacement therapy had greater expression of AHR but not of ARNT in the endometrium and myometrium when compared with women not taking hormones. Leiomyomas expressed significantly less AHR and ARNT mRNA compared with normal myometrium. The ovaries expressed both AHR and ARNT mRNA, and expression was unaffected by age. Endometriotic ovarian cysts expressed more AHR but not more ARNT mRNA compared with healthy ovarian tissue. However, there were no changes in the expression of AHR or ARNT mRNA in ovarian cancer. In conclusion, the female reproductive tract expresses mRNA for the transcription factors AHR and ARNT, and changes in their expression at select target sites in specific pathological conditions such as endometriosis and uterine leiomyomas suggest a potential role for these factors in the pathogenesis of these conditions.

Effect of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on the Expression of Cytochrome P450 1A1, the Aryl Hydrocarbon Receptor, and the Aryl Hydrocarbon Receptor Nuclear Translocator in Rat Brain and Pituitary

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related substances are ubiquitous environmental pollutants causing a wide variety of pathological alterations, with the most severe being progressive anorexia and body weight loss. These features suggest a possible involvement of the nervous system and neuroendocrine-related organs including the pituitary gland. However, so far there is little evidence for direct effects of TCDD on these areas. In the present study, male Sprague–Dawley rats were treated with a single oral dose of TCDD (10 μg/kg) and euthanized 1, 3, or 28 days after treatment. The expression of cytochrome P450 1A1 (CYP1A1), the aryl hydrocarbon receptor (AHR), and the aryl hydrocarbon receptor nuclear translocator (ARNT) were analyzed in different brain regions and pituitaries using semiquantitative RT–PCR and Western blotting. Relative levels of CYP1A1 mRNA and protein were dramatically increased in the pituitary. A significant increase in CYP1A1 mRNA was also detected in all the brain regions examined including olfactory bulb, striatum–caudate, hypothalamus, hippocampus, cortex, cerebellum, and substantia nigra. The increase in the expression was time-dependent with the highest level observed 1 day after TCDD treatment. The AHR and ARNT mRNAs were detected in the same areas but in contrast to CYP1A1 the changes in AHR and ARNT mRNA expression were limited to the 28-day time point. The present results provide evidence for the presence of CYP1A1, AHR, and ARNT in the central nervous system and in the pituitary, suggesting that TCDD may exert a direct effect on these regions.

An uncommon phenotype of poor inducibility of CYP1A1 in human lung is not ascribable to polymorphisms in the AHR, ARNT, or CYP1A1 genes

Cigarette smoking can induce CYP1A1 in the lung. Induction requires the aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins. Lung samples from seven of 75 Finnish patients who smoked until the time of surgery exhibited absent or low levels of CYP1A1 protein, mRNA and enzymatic activity, suggesting that these individuals might be genetically non or poorly inducible for CYP1A1. All seven lung samples expressed normal levels of AHR mRNA and ARNT mRNA, indicating that they did not carry inactivating polymorphisms in the 5' upstream regulatory regions of these genes. Sequencing of cDNAs encompassing the complete coding regions of AHR and ARNT identified a previously known codon 554 polymorphism in AHR, which was present in the homozygous state in one individual. This polymorphism, which leads to an amino acid substitution, has previously been reported either to have no effect or to enhance CYP1A1 induction. Previously unreported silent single nucleotide polymorphisms were identified in codon 44 of AHR and codon 189 of ARNT. 1500 bp of genomic sequence from the 5' upstream regulatory sequence of the CYP1A1 gene was also sequenced in the non-inducible individuals. A nucleotide substitution polymorphism at position -459 was detected in the heterozygous state in two individuals. This polymorphic site does not reside in any known regulatory sequence. The complete CYP1A1 coding sequence and intron/exon boundaries were then sequenced. None of the non or poorly inducible individuals exhibited any polymorphisms, either homozygous or heterozygous compared to representative inducible individuals or the previously published CYP1A1 sequence. Thus, no polymorphisms in the AHR, ARNT or CYP1A1 genes were identified that could be responsible for the non/low inducibility phenotype observed.

Distribution of mRNAs encoding the arylhydrocarbon receptor, arylhydrocarbon receptor nuclear translocator, and arylhydrocarbon receptor nuclear translocator-2 in the rat brain and brainstem.

Dioxin exposure alters a variety of neural functions, most likely through activation of the arylhydrocarbon receptor (AhR) pathway. Many of the adverse effects, including disruption of circadian changes in hormone release and depressed appetite, seem to be mediated by hypothalamic and/or brainstem neurons. However, it is unclear whether these effects are direct or indirect, because there have been no comprehensive studies mapping the expression of components of the AhR pathway in the brain. Therefore, we used a sensitive in situ hybridization histochemical (ISHH) method to map the neural expression of AhR mRNA, as well as those of the mRNAs encoding the AhR dimerization partners, arylhydrocarbon receptor nuclear translocator (ARNT) and ARNT2. We found that AhR, ARNT, and ARNT2 mRNAs were widely distributed throughout the brain and brainstem. There was no neuroanatomic evidence that AhR is preferentially colocalized with ARNT or ARNT2. However, ARNT2, unlike ARNT expression, was relatively high in most regions. The most noteworthy regions in which we found AhR, ARNT, and ARNT2 mRNA were several hypothalamic and brainstem regions involved in the regulation of appetite and circadian rhythms, functions that are disrupted by dioxin exposure. These regions included the arcuate nucleus (Arc), ventromedial hypothalamus (VMH), paraventricular nucleus (PVN), suprachiasmatic nucleus (SCN), nucleus of the solitary tract (NTS), and the dorsal and median raphe nuclei. This neuroanatomic information provides important clues as to the sites and mechanisms underlying the previously unexplained effects of dioxins in the central nervous system.

Expression of the Arylhydrocarbon Receptor and the Arylhydrocarbon Receptor Nuclear Translocator during Early Gestation in the Rabbit Uterus

The arylhydrocarbon receptor (AhR) and the arylhydrocarbon receptor nuclear translocator (ARNT) are members of the PAS gene family mediating toxic effects of xenobiotics such as dioxin and polychlorinated biphenyls. We have analyzed the expression and cellular distribution of rabbit AhR and ARNT mRNA and protein level in the nonpregnant uterus and the pregnant and pseudopregnant uterus at Days 6 to 12 of gestation. In the preimplantation uterus at Day 6 of gestation and in the interimplantation and pseudopregnant uterus at Days 7, 8, 9, and 12 of gestation, low levels of AhR transcripts were detected in the glandular uterine epithelium. Upon attachment of the blastocyst at Day 7 of gestation, a strong expression of AhR and ARNT mRNA was observed in the luminal and glandular epithelium of the antimesometrial uterine compartment. In contrast, AhR and ARNT expression was low in the luminal epithelium of the paraplacental and the mesometrial placental fold. AhR mRNA was also detected in the trophoblast cells. During early placentation at Day 9 of gestation, expression of AhR and ARNT was first observed in the perivascular decidualized stromal cells and, at Day 12, extended to the decidualized stromal cells of the placental bed. Within the placenta, the syncytiotrophoblast expressed only low levels of AhR and ARNT mRNA and no protein. The specific expression patterns of AhR and ARNT during early gestation suggest functional roles for both transcription factors during feto-maternal interactions in the rabbit.

Induction of Arylhydrocarbon Receptor Expression in Embryoblast Cells of Rabbit Preimplantation Blastocysts upon Degeneration of Rauber's Polar Trophoblast

The arylhydrocarbon receptor (AhR) is a ligand-activated transcription factor and mediates carcinogenic, teratogenic, and toxic effects of xenobiotics such as dioxin and coplanar polychlorinated biphenyls. The AhR nuclear translocator (ARNT) is involved in AhR signal transduction. We have analyzed the expression of AhR and ARNT mRNA and AhR protein in Day 3 pc (postcoitum) rabbit morulae and Days 4 and 6 pc blastocysts using RT–PCR, nested PCR, whole mount in situ hybridization, and whole mount immunohistochemistry with subsequent confocal laser scanning analysis. AhR and ARNT transcripts were detected in all stages investigated, indicating coexpression of both transcription factors. AhR protein was localized in the cytoplasm. It was detected in Day 3 pc morulae and in blastocysts. In Day 4 pc blastocysts, only trophoblast cells but not embryoblast cells were immunopositive. However, at Day 6 pc, the embryoblast cells also expressed AhR protein and this expression was correlated with the degeneration of Rauber's trophoblast layer.

Ah Receptor and ARNT Protein and mRNA Concentrations in Rat Prostate: Effects of Stage of Development and 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Treatment

Effects of stage of development and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) protein concentrations in reproductive organs of male rats were determined. AhR protein levels in developing rat ventral and dorsolateral prostate decreased with age, declining approximately 70% between Postnatal Days (PND) 1 and 21. ARNT protein levels also decreased with age in dorsolateral, but not ventral prostate. The developmental decreases in prostatic AhR and ARNT protein were associated with decreases in AhR and ARNT mRNA. AhR and ARNT protein concentrations in fetal urogenital sinus on Gestation Days (GD) 16, 18, and 20 were similar to levels in ventral prostate on PND 7. TCDD exposure of adult male rats (0.2, 1, 5, or 25 μg/kg po, 24 h) decreased AhR but not ARNT protein in ventral and dorsolateral prostate, vas deferens, and epididymis.In uteroand lactational TCDD exposure (1.0 μg/kg dam po, GD 15) did not alter ARNT levels but reduced prostatic AhR protein levels on PND 7 and delayed the developmental decrease in AhR protein in ventral and dorsolateral prostate. Finally, pretreatment of rat pups for 24 h with TCDD (5 μg/kg ip) down-regulated prostatic AhR protein on PND 7, but not on PND 1. Thus, prostatic AhR and ARNT protein and mRNA levels are regulated with age, whereas only AhR protein concentration is altered by TCDD exposure. Becausein uteroand lactational TCDD exposure only decreased prostatic AhR on PND 7, it is unlikely that down-regulation of AhR is the mechanism by which perinatal TCDD exposure impairs prostate development.