Aryl Hydrocarbon Receptor Repressor Research Articles

Transcriptomic analysis of Anabas testudineus and its defensive mechanisms in response to persistent organic pollutants exposure

The freshwater climbing perch (Anabas testudineus) can tolerate water environments contaminated with persistent organic pollutants (POPs). The mechanisms underlying this tolerance are unknown. We used de novo transcriptomic analysis to investigate the defensive mechanisms of A. testudineus against POPs based on its genetic features and biological responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. Our results revealed a specific expansion of cytochrome P450 (CYP) 3A subfamily, which may be involved in the elimination of certain POPs. In xenobiotic responses, the aryl-hydrocarbon receptor (AhR) pathway represents a critical signaling mechanism, and we characterized four AhR and two AhR nuclear translocator homologs and one AhR repressor (AhRR) gene in A. testudineus. TCDD-induced AhRR and CYP1A mRNA upregulation suggests that negative-feedback regulation of AhR signaling through AhRR helps avoid excessive xenobiotic responses. Furthermore, liver and gill transcriptomic profiles were markedly altered after TCDD exposure, with some of the altered genes being related to common defensive responses reported in other species. Based on the newly identified TCDD-altered genes, several A. testudineus-specific responses are proposed, such as enhanced fatty acid β-oxidation. The genetic features of CYP3A subfamily and AhR pathway and the TCDD-induced defensive biological processes elucidated here enhance our understanding of A. testudineus defensive responses against POPs.

The effect of body mass index on smoking behaviour and nicotine metabolism: a Mendelian randomization study.

Given clear evidence that smoking lowers weight, it is possible that individuals with higher body mass index (BMI) smoke in order to lose or maintain their weight. We performed Mendelian randomization (MR) analyses of the effects of BMI on smoking behaviour in UK Biobank and the Tobacco and Genetics Consortium genome-wide association study (GWAS), on cotinine levels and nicotine metabolite ratio (NMR) in published GWAS and on DNA methylation in the Avon Longitudinal Study of Parents and Children. Our results indicate that higher BMI causally influences lifetime smoking, smoking initiation, smoking heaviness and also DNA methylation at the aryl-hydrocarbon receptor repressor (AHRR) locus, but we do not see evidence for an effect on smoking cessation. While there is no strong evidence that BMI causally influences cotinine levels, suggestive evidence for a negative causal influence on NMR may explain this. There is a causal effect of BMI on smoking, but the relationship is likely to be complex due to opposing effects on behaviour and metabolism.

Association of the human aryl hydrocarbon receptor repressor (AhRR)-c.565C>G transversion with male infertility: A case-control study from Iran.

Dioxins (eg, 2,3,7,8-tetrachlorodibenzo-p-dioxin/TCDD), as environmental endocrine disruptors and toxic carcinogens, can affectmale reproductive health. The influence of dioxins is mediated via the aryl hydrocarbon receptor (AhR) pathway and its repressor (AhRR). In this study, we investigated the association of AhRR-c.565C>G transversion polymorphism with male infertility. In a hospital-based case-control study, 221 semen samples (111 infertile and 110 healthy controls) based onWorld Health Organization guidelines were collected from in vitro fertilization centers of Babol, Iran. The AhRR-c.565C>G (rs2292596) polymorphism was genotyped using a polymerase chain reaction-restriction fragment length polymorphism analysis. The difference in the allele frequency of AhRR-c.565C>G transversion polymorphism did not reach a significant level. The genotype frequency was statistically significantly different between fertile and infertile men. We found that polymorphism rs2292596 (Pro185Ala) was statistically significantly associated with the risk of male infertility. In addition, the statistical difference became more significant when the frequency was compared between the Pro/Pro genotype and the Pro/Ala plus Ala/Ala genotype. The 185 Pro wild-type alleles of AhRR may be associated with the risk of male infertility. The proallele also may diminish inhibition of AhR-mediated signaling of exposure to environmental pollutants.

Abstract 16728: Cigarette Smoke Impairs the Anti-Oxidative Response by Reducing Heme Levels and Inducing Bach1

Introduction: Cigarette smoke is a major risk factor of cardiovascular diseases. Heme regulates the activity of enzymes and transcription factors including Bach1. Bach1 competes with Nrf2 transcriptional activity and inhibits the expression of anti-oxidative enzymes. However, the molecular mechanism of Bach1 activation and its impact on the anti-oxidative response after exposure to cigarette smoke is not clear. Hypothesis: We aim to understand the mechanism through which cigarette smoke impairs the anti-oxidative response. Methods: Primary human umbilical vein endothelial cells (HUVEC) were exposed to a cigarette smoke extract (CSE) (3 mg/ml nicotine) under static and unidirectional shear stress conditions. Shear stress was induced using the ibidi pump system and cone-and-plate viscometer. Llevels of reduced glutathione (GSH) were measured using the fluorescent probe o-phthalaldehyde. Heme levels were measured after oxalic acid extraction. Gene and miR expression was quantified by RT-qPCR. Immunofluorescence imaging was performed on fixed HUVEC. MiR-125b overexpression was achieved by transfecting mimics. Inhibition of Bach1 was done using siRNA transfection. Apoptosis was assessed using the caspase 3/7 assay. Human umbilical veins as an ex vivo model were exposed to CSE under static conditions. Results: Exposure to CSE induced the expression of BACH1 (1.90±0.18; p<0.05) and reduced total heme levels (0.30±0.04; p<0.001) in HUVEC. It also reduced miR-125b expression in HUVEC (0.39±0.09; p<0.001) and umbilical veins (0.72±0.12; p<0.05), whereas the expression of miR-125b target AhR repressor ( AHRR ) increased (14.93±4.08; p<0.05). Inhibiting BACH1 rescued miR-125b expression (1.02±0.45; p>0.05) and reduced AHRR expression (0.66±0.12; P<0.05). Acute exposure to CSE led to a high oxidative stress load. This observation was supported by a 2-fold reduction in glutathione levels, an increase in nuclear:cytosolic ratio of the upstream transcription factor Nrf2 (1.33±0.05; p<0.01) and an increase in expression of anti-oxidative HMOX1 (18.18±1.8; p<0.001) and NQO1 (16.12±1.99; p<0.001). CSE-induced apoptosis (1.23±0.04; p<0.001) was reversible by pre-conditioning with 1 mM N-acetyl cysteine (0.99±0.02; p>0.05) or overexpression of miR-125b (0.99±0.07; p>0.05). Conclusions: These results show that cigarette smoke-induced Bach1 impairs the anti-oxidative response through inhibiting miR-125b in the human endothelium. Reduced heme levels could be involved in the upregulation of Bach1 activity.

1 - Cigarette smoke impairs the anti-oxidative response by reducing heme levels and inducing Bach1

Introduction Cigarette smoke is a major risk factor of cardiovascular diseases. Heme regulates the activity of enzymes and transcription factors including Bach1. Bach1 competes with Nrf2 transcriptional activity and inhibits the expression of anti-oxidative enzymes. However, the molecular mechanism of Bach1 activation and its impact on the anti-oxidative response after exposure to cigarette smoke is not clear. We aim to understand the mechanism through which cigarette smoke impairs the anti-oxidative response. Methods Primary human umbilical vein endothelial cells (HUVEC) were exposed to a cigarette smoke extract (CSE) under static and unidirectional shear stress conditions. Shear stress was induced using the ibidi pump system and cone-and-plate viscometer. Levels of reduced glutathione (GSH) were measured using the fluorescent probe o-phthalaldehyde. Heme levels were measured after oxalic acid extraction. Gene and miR expression was quantified by RT-qPCR. Immunofluorescence imaging was performed on fixed HUVEC. MiR-125b overexpression was achieved by transfecting mimics. Inhibition of Bach1 was done using siRNA transfection. Apoptosis was assessed using caspase 3/7 activity. Human umbilical veins as an ex vivo model were exposed to CSE under static conditions. Results Exposure to CSE induced the expression of BACH1 and reduced total heme levels in HUVEC. It also reduced miR-125b expression in HUVEC and umbilical veins, whereas the expression of miR-125b target AhR repressor (AHRR) increased. Inhibiting BACH1 increased miR-125b expression and reduced AHRR expression. Acute exposure to CSE led to a high oxidative stress load. This observation was supported by a decrease in glutathione levels, an increase in nuclear:cytosolic ratio of the upstream transcription factor Nrf2 and an increase in expression of HMOX1 and NQO1. CSE-induced apoptosis was reversible by preconditioning with N-acetyl cysteine or overexpression of miR-125b. Conclusions cigarette smoke-induced Bach1 impairs the anti-oxidative response through inhibiting miR-125b. Reduced heme levels could be involved in the upregulation of Bach1 activity.

The increase of interfollicular epidermal stem cells and regulation of aryl hydrocarbon receptor and its repressors in the skin through hydroporation with anti-aging cocktail.

Hydroporation is a procedure that involves a subsonic flow of air and microdroplets into the skin. We previously reported that hydroporation treatment with a cocktail solution containing copper-glycyl-L-histidyl-L-lysyl, oligo hyaluronic acid, rhodiola extract, tranexamic acid, and β-glucan yielded positive effects on skin aging. The aim of this study was to evaluate the effects of hydroporation with anti-aging cocktail on interfollicular epidermal stem cells (IFESCs) and expression of aryl hydrocarbon receptor (AhR)/AhR repressor (AhRR) in the skin. Skin samples from six volunteers who were treated with hydroporation were analyzed via confocal microscopic examination. Markers for dermal matrix (procollagen type I and fibrillin-1) and basement membrane (type IV collagen and integrin α6) were increased after treatment. Moreover, there was a significant increase in the expression level of histone deacetylase 1-positive/p63-negative basal cells, which we previously reported as interfollicular epidermal stem cells. The expression level of AhR was significantly decreased, whereas that of AhRR was increased. This indicates an alteration in the interaction between the skin and environment posttreatment. Anti-aging hydroporation treatment recovered the stem cell potential of basal cells. Moreover, this treatment decreased AhR and increased AhRR in the skin, which may protect the skin from the harmful environment.

Abstract A08: Widespread tobacco smoking-associated changes in DNA methylation and gene expression in lung tissue of smokers

Abstract Significance: Epigenome-wide association studies (EWAS) have identified thousands of CpGs showing smoking-associated DNA methylation changes in blood cell DNA of smokers. Several of these CpGs are strongly associated with lung cancer risk. Until recently, the relationship of such DNA methylation changes in the blood and epigenetic alterations in the lung, as well as their molecular link to lung cancer development, remained poorly understood. Using a limited EWAS of 237 lung tissue samples, we recently identified seven smoking-associated hypomethylated CpGs in lung tissue, five of which had been previously reported in blood, and one of which had been strongly associated with lung cancer risk. We showed that the latter CpG flanked a smoking-inducible enhancer element driving expression of a xenobiotic response gene. Here we expand these observations to identify tobacco-affected regulatory elements implicated in lung cancer. Methods: We performed an EWAS using Inifinium HumanMethylation450K BeadChip data from The Cancer Genome Atlas (TCGA) corresponding to 393 eligible lung adenocarcinoma tumor samples. We replicated our findings in 27 adjacent normal lung tissue samples, then examined epigenetic enhancer marks near the smoking-associated altered CpGs using epigenomic data from primary lung cells and lung adenocarcinoma cell lines. We next identified potential responsive target genes of these enhancers by examining smoking-associated changes in gene expression on the 1 megabase flanks of the enhancers. Results: 37,372 CpGs were significantly differentially methylated with respect to smoking in TCGA adenocarcinoma tumor samples after Benjamini Hochberg correction (p<0.05). 2,941 probes replicated at a nominal level in the adjacent normal samples (p<0.05), including 145 probes previously associated with smoking in at least one blood-based EWAS. 1,150 of the 2,941 replicated probes were located within 2 kb of a likely functional region carrying a histone mark of active enhancers in primary lung cells and/or lung cancer cell lines. 949 of these were within 1 Mb of a gene showing a significant, smoking-associated expression difference. One of the top-ranked CpGs we identified was among the 10 most-replicated CpGs in blood-based smoking EWAS, and was linked to smoking-induced upregulation of the aryl hydrocarbon receptor repressor (AHRR) gene, which is involved in the cellular xenobiotic response and has been implicated in lung cancer risk. Conclusion: Many smoking-associated methylation changes in lung tissue are located in the vicinity of epigenetic regulators in lung tissue, and could be driving expression of smoking-associated genes that may affect lung cancer risk. Funding: R01 HL114094 to IALO, P30 CA014089, USC Provost’s fellowship and Roy E. Thomas Foundation Graduate Scholarship to DJM. Citation Format: Daniel J. Mullen, T. Ryan Stueve, Chunli Yan, Kimberly D. Siegmund, Ite A. Laird-Offringa. Widespread tobacco smoking-associated changes in DNA methylation and gene expression in lung tissue of smokers [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A08.

Association between polycystic ovary syndrome and the polymorphisms of aryl hydrocarbon receptor repressor, glutathione-s-transferase T1, and glutathione-S-transferase M1 genes

Association between polycystic ovary syndrome and the polymorphisms of aryl hydrocarbon receptor repressor, glutathione-s-transferase T1, and glutathione-S-transferase M1 genes

Toxicology tailored low density oligonucleotide microarray for the thicklip grey mullets (Chelon labrosus): Biomarker gene transcription profile after caging in a polluted harbour

In aquatic organisms inhabiting polluted waters genes are activated to build an adaptive/compensatory defence against the possible effects of pollutants. Such responses can be used as biomarkers of exposure to chemical compounds, outlining the molecular mechanisms activated under specific pollution scenarios. With the aim of exploiting such approach in environmental health assessment, toxicologically relevant gene fragments were sequenced in the thicklip grey mullet (Chelon labrosus) and a toxicologically tailored low-density (160 genes) oligonucleotide microarray was customised. The tool was validated comparing organ/sex specific gene expression profiles and characterising responses under laboratory exposure to model chemicals. Finally, juvenile mullets were caged in a polluted harbour and hepatic gene expression profiles analysed after 5 and 21 days of deployment. Cages were deployed in the inner (IH) and outer (OH) Pasaia harbour, Bay of Biscay. Mussels (Mytilus galloprovincialis) were also caged as biological matrix for chemical bioaccumulation analysis and stress biomarkers measurements. Slightly higher concentrations of chemicals (metals, tributyltin, PAHs, phthalates) were quantified in IH than in OH, fish bile metabolites also revealing higher availability of PAHs in IH. Lysosome membrane stability in mussels was reduced, indicating stress condition in both sites. The developed microarray discriminated mullets showing distinctive expression profiles depending on site and deployment time. Genes related to immune and hypoxia responses were regulated comparing IH and OH at day 5. Phase I and II biotransformation genes, such as cyp2, cyp3 and ugt, were up-regulated in IH, together with the aryl hydrocarbon receptor 2 (ahr2) and the ahr repressor. Similarly, TBT-binding proteins and genes involved in lipid metabolism (pparγ, cyp7) were up-regulated with deployment time. Even if nowadays higher throughput approaches for gene expression analyses are available, the developed mullet tool constitutes a comprehensive tool to assess molecular responses of mullets exposed to pollutants, although it remains to be explored whether it can be applied to assess pollutant exposure in active pollution monitorings and in environmental health assessment.

RNA-Seq analysis of transcriptome responses in Atlantic cod (Gadus morhua) precision-cut liver slices exposed to benzo[a]pyrene and 17α-ethynylestradiol

Polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) that activate the aryl hydrocarbon receptor (Ahr) pathway, and endocrine disruptors acting through the estrogen receptor pathway are among environmental pollutants of major concern. In this work, we exposed Atlantic cod (Gadus morhua) precision-cut liver slices (PCLS) to BaP (10 nM and 1000 nM), ethynylestradiol (EE2) (10 nM and 1000 nM), and equimolar mixtures of BaP and EE2 (10 nM and 1000 nM) for 48 h, and performed RNA-Seq based transcriptome mapping followed by systematic bioinformatics analyses. Our gene expression analysis showed that several genes were differentially expressed in response to BaP and EE2 treatments in PCLS. Strong up-regulation of genes coding for the cytochrome P450 1a (Cyp1a) enzyme and the Ahr repressor (Ahrrb) was observed in BaP treated PCLS. EE2 treatment of liver slices strongly up-regulated genes coding for precursors of vitellogenin (Vtg) and eggshell zona pellucida (Zp) proteins. As expected, pathway enrichment and network analysis showed that the Ahr and estrogen receptor pathways are among the top affected by BaP and EE2 treatments, respectively. Interestingly, two genes coding for fibroblast growth factor 3 (Fgf3) and fibroblast growth factor 4 (Fgf4) were up-regulated by EE2 in this study. To our knowledge, the fgf3 and fgf4 genes have not previously been described in relation to estrogen signaling in fish liver, and these results suggest the modulation of the FGF signaling pathway by estrogens in fish. The signature expression profiles of top differentially expressed genes in response to the single compound (BaP or EE2) treatment were generally maintained in the expression responses to the equimolar binary mixtures. However, in the mixture-treated groups, BaP appeared to have anti-estrogenic effects as observed by lower number of differentially expressed putative EE2 responsive genes. Our in-depth quantitative analysis of changes in liver transcriptome in response to BaP and EE2, using PCLS tissue culture provides further mechanistic insights into effects of the compounds. Moreover, the analyses demonstrate the usefulness of PCLS in cod for omics experiments.

Towards Resolving the Pro- and Anti-Tumor Effects of the Aryl Hydrocarbon Receptor.

We have postulated that the aryl hydrocarbon receptor (AHR) drives the later, more lethal stages of some cancers when chronically activated by endogenous ligands. However, other studies have suggested that, under some circumstances, the AHR can oppose tumor aggression. Resolving this apparent contradiction is critical to the design of AHR-targeted cancer therapeutics. Molecular (siRNA, shRNA, AHR repressor, CRISPR-Cas9) and pharmacological (AHR inhibitors) approaches were used to confirm the hypothesis that AHR inhibition reduces human cancer cell invasion (irregular colony growth in 3D Matrigel cultures and Boyden chambers), migration (scratch wound assay) and metastasis (human cancer cell xenografts in zebrafish). Furthermore, these assays were used for a head-to-head comparison between AHR antagonists and agonists. AHR inhibition or knockdown/knockout consistently reduced human ER−/PR−/Her2− and inflammatory breast cancer cell invasion, migration, and metastasis. This was associated with a decrease in invasion-associated genes (e.g., Fibronectin, VCAM1, Thrombospondin, MMP1) and an increase in CDH1/E-cadherin, previously associated with decreased tumor aggression. Paradoxically, AHR agonists (2,3,7,8-tetrachlorodibenzo-p-dioxin and/or 3,3′-diindolylmethane) similarly inhibited irregular colony formation in Matrigel and blocked metastasis in vivo but accelerated migration. These data demonstrate the complexity of modulating AHR activity in cancer while suggesting that AHR inhibitors, and, under some circumstances, AHR agonists, may be useful as cancer therapeutics.

Ambient particulate matter activates the aryl hydrocarbon receptor in dendritic cells and enhances Th17 polarization

The objective of this study was to explore the role of the aryl hydrocarbon receptor (AhR) in ambient particulate matter (PM)-mediated activation of dendritic cells (DCs) and Th17-immune responses in vitro. To assess the potential role of the AhR in PM-mediated activation of DCs, co-stimulation, and cytokine expression, bone marrow (BM)-derived macrophages and DCs from C57BL/6 wildtype or AhR knockout (AhR−/−) mice were treated with PM. Th17 differentiation was assessed via co-cultures of wildtype or AhR−/− BMDCs with autologous naive T cells. PM2.5 significantly induced AhR DNA binding activity to dioxin responsive elements (DRE) and expression of the AhR repressor (AhRR), cytochrome P450 (CYP) 1A1, and CYP1B1, indicating activation of the AhR. In activated (OVA sensitized) BMDCs, PM2.5 induced interleukin (IL)-1β, CD80, CD86, and MHC class II, suggesting enhanced DC activation, co-stimulation, and antigen presentation; responses that were abolished in AhR deficient DCs. DC-T cell co-cultures treated with PM and lipopolysaccharide (LPS) led to elevated IL-17A and IL-22 expression at the mRNA level, which is mediated by the AhR. PM-treated DCs were essential in endowing T cells with a Th17-phenotype, which was associated with enhanced expression of MHC class II and cyclooxygenase (COX)-2. In conclusion, PM enhances DC activation that primes naive T cell differentiation towards a Th17-like phenotype in an AhR-dependent manner.

Identification of Smoking-Associated Differentially Methylated Regions Using Reduced Representation Bisulfite Sequencing and Cell type-Specific Enhancer Activation and Gene Expression.

Background:Cigarette smoke is a causal factor in cancers and cardiovascular disease. Smoking-associated differentially methylated regions (SM-DMRs) have been observed in disease studies, but the causal link between altered DNA methylation and transcriptional change is obscure.Objective:Our objectives were to finely resolve SM-DMRs and to interrogate the mechanistic link between SM-DMRs and altered transcription of enhancer noncoding RNA (eRNA) and mRNA in human circulating monocytes.Method:We integrated SM-DMRs identified by reduced representation bisulfite sequencing (RRBS) of circulating monocyte DNA collected from two independent human studies [ from Clinical Research Unit (CRU) and from the Multi-Ethnic Study of Atherosclerosis (MESA), about half of whom were active smokers] with gene expression for protein-coding genes and noncoding RNAs measured by RT-PCR or RNA sequencing. Candidate SM-DMRs were compared with RRBS of purified T cells, T cells, granulocytes, B cells, and NK cells ( females, CRU). DMRs were validated using pyrosequencing or bisulfite amplicon sequencing in up to 85 CRU volunteers, who also provided saliva DNA.Results:RRBS identified monocyte SM-DMRs frequently located in putative gene regulatory regions. The most significant monocyte DMR occurred at a poised enhancer in the aryl-hydrocarbon receptor repressor gene (AHRR) and it was also detected in both granulocytes and saliva DNA. To our knowledge, we identify for the first time that SM-DMRs in or near AHRR, C5orf55-EXOC-AS, and SASH1 were associated with increased noncoding eRNA as well as mRNA in monocytes. Functionally, the AHRR SM-DMR appeared to up-regulate AHRR mRNA through activating the AHRR enhancer, as suggested by increased eRNA in the monocytes, but not granulocytes, from smokers compared with nonsmokers.Conclusions:Our findings suggest that AHRR SM-DMR up-regulates AHRR mRNA in a monocyte-specific manner by activating the AHRR enhancer. Cell type–specific activation of enhancers at SM-DMRs may represent a mechanism driving smoking-related disease. https://doi.org/10.1289/EHP2395

Mechanistic Role of Cytochrome P450 1B1 in Hyperoxic Lung Injury

Supplemental oxygen is often used to assist those with respiratory dysfunction, including adults with acute respiratory distress syndrome (ARDS) and bronchopulmonary dysplasia (BPD) in preterm infants. Experimental evidence has shown that increases in oxygen levels can create a hyperoxic environment that is capable of exacerbating existing pulmonary injury as well as independently inducing injury due to increases in reactive oxygen species (ROS) and oxidative stress. Our group has examined the role of cytochrome P450 (CYP) enzymes in hyperoxic toxicity because of their possible role in the generation of ROS. We have previously found that CYP1A1/1A2 as well as the aryl hydrocarbon receptor (AHR), protect against hyperoxic toxicity. We have also found that CYP1B1 may, in fact, contribute to hyperoxic toxicity in vitro and in Cyp1b1‐null mice. Further, we found that Cyp1b1‐null mice exposed to hyperoxia have decreased markers of oxidative stress and increased CYP1A gene expression compared to WT mice. In this study, we hypothesized that Cyp1b1‐null mice would be less susceptible to hyperoxic lung injury compared to WT mice and that CYP1A mechanistically contributes to the attenuated phenotype of Cyp1b1‐null mice. To test this hypothesis, we exposed male and female C57BL/6J (WT), Cyp1b1‐null, Cyp1a1/1b1‐null (DKO), and Cyp1a1/1a2/1b1‐null (TKO) mice to hyperoxia (>95% O2) for 24–68 hours. Lung weight measurements were used to assess pulmonary injury and edema and qPCR was used to measure RNA. We found that all mice lacking CYP1B1, including DKO and TKO mice, had reduced pulmonary injury as indicated by reduced lung weights in male (P<0.01) and female (P<0.05) mice after 68 hours of hyperoxia. CYP1B1 gene expression was induced (P<0.01) in WT mice during hyperoxia exposure, and CYP1A1 gene expression was increased in Cyp1b1‐null mice (P=0.01). NQO1 gene expression was significantly induced by hyperoxia (P<0.05) in all groups with higher expression in TKO mice at room air (P<0.001) and early phase of hyperoxia (P<0.05). We found that pulmonary AHR repressor (AHRR) expression was increased in the DKO and TKO (P<0.05) mice after hyperoxia exposure. Our results suggest that increased AHR‐dependent transcription, as evidenced by enhanced CYP1A1 and AHRR gene expression, may play a role in attenuating lung injury in mice lacking CYP1B1 and needs to be elucidated further. Future studies will thoroughly characterize the effect of CYP1B1 on oxidative stress in these animals and determine the viability of CYP1B1‐directed therapeutics to treat or prevent ARDS/BPD.Support or Funding InformationResearch reported in this publication was supported by the National Institutes of Health under award number R01ES019689, R01ES009132, R01HL12516, R01HL129794, and R01HL087174 to B.M. and T32GM088129 to A.V. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Suppressive effects of aryl-hydrocarbon receptor repressor on adipocyte differentiation in 3T3-L1 cells

The aryl-hydrocarbon receptor repressor (AhRR) negatively regulates aryl-hydrocarbon receptor (AhR) signaling via its inhibitory transactivation. AhR is well known to suppress adipocyte differentiation, but the function of AhRR during adipogenesis is unclear. The purpose of this study was to investigate the role of AhRR in adipocyte differentiation using 3T3-L1 cells. During the early phase of differentiation, AhRR expression was transiently induced, but throughout the entire differentiation process, low levels of AhR expression were maintained. AhRR knockdown significantly increased not only glycerol-3-phosphate dehydrogenase (GPDH) activity but also lipid accumulation inside the cells. AhRR overexpression clearly reduced GPDH activity and lipid accumulation, indicating that AhRR upregulation during the early stage of adipogenesis suppresses adipocyte differentiation. Since AhRR knockdown increases the expression and activity of peroxisome proliferator-activated receptor γ (PPARγ), AhRR negatively regulates PPARγ during adipogenesis. In summary, similar to AhR, AhRR acts as an inhibitor of adipocyte differentiation. In addition to controlling the negative feedback loop of AhR, AhRR might be involved in other functions, especially in adipocyte differentiation processes.

AHRR hypomethylation, lung function, lung function decline and respiratory symptoms.

Epigenome-wide association studies have shown a consistent association between smoking exposure and hypomethylation in the aryl hydrocarbon receptor repressor (AHRR) gene (cg05575921). We tested the hypothesis that AHRR hypomethylation is associated with low lung function, steeper lung function decline, and respiratory symptoms in the general population.AHRR methylation extent was measured in 9113 individuals from the 1991-1994 examination of the Copenhagen City Heart Study, using bisulfite-treated leukocyte DNA. Spirometry at the time of blood sampling was available for all individuals. Lung function was measured again for 4532 of these individuals in 2001-2003.Cross-sectionally, a 10% lower methylation extent was associated with a 0.2 z-score (95% CI 0.1-0.2) lower forced expiratory volume in 1 s (FEV1) after multivariable adjustment including smoking. Hypomethylation was also associated with a lower z-score for both forced vital capacity (FVC) and FEV1/FVC. In prospective analyses, individuals in the lowest versus highest tertile of methylation extent had a steeper decline in FEV1/height3 (p for examination×methylation interaction=0.003) and FVC/height3 (p=0.01), but not FEV1/FVC (p=0.08). Multivariable-adjusted odds ratios per 10% lower methylation extent were 1.31 (95% CI 1.18-1.45) for chronic bronchitis and 1.21 (95% CI 1.13-1.30) for any respiratory symptoms.AHRR hypomethylation was associated with low lung function, steeper lung function decline, and respiratory symptoms.

Urinary 1-hydroxypyrene and smoking are determinants of LINE-1 and AhRR promoter methylation in coke oven workers

Coke oven emissions (COE) containing polycyclic aromatic hydrocarbons (PAHs) are predominant toxic constituents of particulate air pollution that have been linked to increased risk of lung cancer. Aberrant DNA methylation is one of the best known epigenetic changes in human cancers and healthy subjects exposed to carcinogens. The purpose of this study is to explore the factors influencing the methylation of long interspersed nuclear element-1 (LINE-1) and aryl-hydrocarbon receptor repressor (AhRR) in coke oven workers. The study population is composed by coke oven workers (348) and water treatment workers (131). And their urinary PAH metabolites were analyzed by high performance liquid chromatography; DNA methylation were measured by pyrosequencing. The urinary PAHs metabolites were significantly elevated in coke oven workers (P < 0.01). The results from multivariate logistic regression analysis showed that a high level of urinary 1-hydroxypyrene was associated with a significantly increased risk of hypomethylation of LINE-1 (OR: 1.80; 95% CI: 1.25, 2.60), and heavy smoking was associated with a significantly increased risk of hypomethylation of AhRR (OR: 1.44; 95% CI: 1.04, 2.00). Our findings demonstrate that urinary 1-hydroxypyrene may be a useful biomarker for evaluating the role of PAHs exposure on hypomethylation of LINE-1 among coke oven workers and that smoking may be an important factor affecting hypomethylation of AhRR.

High AHR expression in breast tumors correlates with expression of genes from several signaling pathways namely inflammation and endogenous tryptophan metabolism.

Increasing epidemiological and animal experimental data provide substantial support for the role of aryl hydrocarbon receptor (AhR) in mammary tumorigenesis. The effects of AhR have been clearly demonstrated in rodent models of breast carcinogenesis and in several established human breast cancer cell lines following exposure to AhR ligands or AhR overexpression. However, relatively little is known about the role of AhR in human breast cancers. AhR has always been considered to be a regulator of toxic and carcinogenic responses to environmental contaminants such as TCDD (dioxin) and benzo[a]pyrene (BaP). The aim of this study was to identify the type of breast tumors (ERα-positive or ERα-negative) that express AHR and how AhR affects human tumorigenesis. The levels of AHR, AHR nuclear translocator (ARNT) and AHR repressor (AHRR) mRNA expression were analyzed in a cohort of 439 breast tumors, demonstrating a weak association between high AHR expression and age greater than fifty years and ERα-negative status, and HR-/ERBB2 breast cancer subtypes. AHRR mRNA expression was associated with metastasis-free survival, while AHR mRNA expression was not. Immunohistochemistry revealed the presence of AhR protein in both tumor cells (nucleus and/or cytoplasm) and the tumor microenvironment (including endothelial cells and lymphocytes). High AHR expression was correlated with high expression of several genes involved in signaling pathways related to inflammation (IL1B, IL6, TNF, IL8 and CXCR4), metabolism (IDO1 and TDO2 from the kynurenine pathway), invasion (MMP1, MMP2 and PLAU), and IGF signaling (IGF2R, IGF1R and TGFB1). Two well-known ligands for AHR (TCDD and BaP) induced mRNA expression of IL1B and IL6 in an ERα-negative breast tumor cell line. The breast cancer ER status likely influences AhR activity involved in these signaling pathways. The mechanisms involved in AhR activation and target gene expression in breast cancers are also discussed.

Involvement of Aryl Hydrocarbon Receptor and Aryl Hydrocarbon Receptor Repressor in Helicobacter Pylori-related Gastric Pathogenesis.

Background: Persistent Helicobacter pylori (H. pylori) infection leads to various gastric diseases. Multiple studies have demonstrated that aryl hydrocarbon receptor (AHR) plays roles in the antibacterial response and aryl hydrocarbon receptor repressor (AHRR) is downregulated in stomach cancer. However, the role of AHR or AHRR in H. pylori-related gastric diseases remains unclear.Aims: To investigate whether AHR or AHRR is involved in H. pylori-related gastric diseases.Methods: Patients with gastritis or gastric adenocarcinoma were enrolled randomly, and gastric tissue specimens were diagnosed pathologically. AHR, AHRR, and H. pylori infection status in tissues were detected by immunohistochemistry. Human gastric cells were cocultured with H. pylori. siRNAs were used to silence AHR or AHRR, and a C57bl/6 mouse model colonized by H. pylori was established. Protein expression was determined by western blotting analysis, and TNF, IL-8 and IL-1β in cell supernatants were measured by ELISA.Results: AHR and AHRR were expressed in gastritis tissues and gastric cancer tissues without H. pylori infection, and principally located in the cytoplasm and nucleus. AHR expression was significantly correlated with AHRR expression in gastric tissues without H. pylori infection (P=0.008). However, their expressions were negatively correlated with H. pylori infection status. H. pylori coculture inhibited AHR and AHRR expression in stomach mucosa in vitro and in vivo. Gastric cells produced more TNF, IL-8 and IL-1β when AHR or AHRR was silenced.Conclusions: This preliminary study indicates that AHR and AHRR may be involved in H. pylori-related gastric pathogenesis, and helps toward understanding of inflammation-initiated carcinogenesis of gastric cancer.

The transcription factors GATA2 and microphthalmia-associated transcription factor regulate Hdc gene expression in mast cells and are required for IgE/mast cell–mediated anaphylaxis

Histamine is a critical mediator of IgE/mast cell-mediated anaphylaxis. Histamine is synthesized by decarboxylating the amino acid histidine, a reaction catalyzed by the histidine decarboxylase (Hdc) gene-encoded enzyme HDC. However, regulation of the Hdc gene in mast cells is poorly understood. We sought to investigate the invivo regulation of IgE/mast cell-mediated anaphylaxis by the transcription factors GATA2 and microphthalmia-associated transcription factor (MITF) and the mechanisms by which GATA2 and MITF regulate Hdc gene expression in mouse and human mast cells. Mice deficient in the transcription factors Gata2, aryl hydrocarbon receptor (Ahr), aryl hydrocarbon receptor repressor (Ahrr), or basic helix-loop-helix family member E40 (Bhlhe40) were assessed for anaphylactic reactions. Chromatin immunoprecipitation sequencing analysis identified putative Hdc enhancers. Luciferase reporter transcription assay confirmed enhancer activities of putative enhancers in the Hdc gene. The short hairpin RNA knockdown approach was used to determine the role of MITF in regulating mouse and human HDC gene expression. Connective tissue mast cell-specific Gata2-deficient mice did not have IgE/mast cell-mediated anaphylaxis. GATA2 induced the expression of Mitf, Ahr, Ahrr, and Bhlhe40 in mast cells. MITF, but not AHR, AHRR, or BHLHE40, was required for anaphylaxis. MITF bound to an enhancer located 8.8 kb upstream of the transcription start site of the Hdc gene and directed enhancer activity. MITF overexpression largely restored Hdc gene expression in the Gata2-deficient mast cells. In the human mast cell line LAD2, MITF was required for the HDC gene expression and histamine synthesis. The transcription factors GATA2 and MITF regulate Hdc gene expression in mast cells and are required for IgE/mast cell-mediated anaphylaxis.