Repression of the aryl-hydrocarbon receptor prevents oxidative stress and ferroptosis of intestinal intraepithelial lymphocytes.

The aryl-hydrocarbon receptor (AHR) is a ligand-activated transcription factor that buoys intestinal immune responses. AHR induces its own negative regulator, the AHR repressor (AHRR). Here, we show that AHRR is vital to sustaining intestinal intraepithelial lymphocytes (IELs). AHRR deficiency reduced IEL representation in a cell-intrinsic fashion. Single-cell RNA sequencing revealed an oxidative stress profile in Ahrr-/- IELs. AHRR deficiency unleashed AHR-induced expression of CYP1A1, a monooxygenase that generates reactive oxygen species, increasing redox imbalance, lipid peroxidation, and ferroptosis in Ahrr-/- IELs. Dietary supplementation with selenium or vitamin E to restore redox homeostasis rescued Ahrr-/- IELs. Loss of IELs in Ahrr-/- mice caused susceptibility to Clostridium difficile infection and dextran sodium-sulfate-induced colitis. Inflamed tissue of inflammatory bowel disease patients showed reduced Ahrr expression that may contribute to disease. We conclude that AHR signaling must be tightly regulated to prevent oxidative stress and ferroptosis of IELs and to preserve intestinal immune responses.

Silencing of aryl hydrocarbon receptor repressor restrains Th17 cell immunity in autoimmune hepatitis.

Th17-cells play a key role in the pathogenesis of autoimmune hepatitis (AIH). Dysregulation of Th17-cells in AIH is linked to defective response to aryl-hydrocarbon-receptor (AhR) activation. AhR modulates adaptive immunity and is regulated by aryl-hydrocarbon-receptor-repressor (AHRR), which inhibits AhR transcriptional activity. In this study, we investigated whether defective Th17-cell response to AhR derives from aberrant AHRR regulation in AIH. Th17-cells, obtained from the peripheral blood of AIH patients (n = 30) and healthy controls (n = 30) were exposed to AhR endogenous ligands, and their response assessed in the absence or presence of AHRR silencing. Therapeutic effects of AHRR blockade were tested in a model of Concanavalin-A (Con-A)-induced liver injury in humanized mice. AHRR was markedly upregulated in AIH Th17-cells, following exposure to l-kynurenine, an AhR endogenous ligand. In patients, silencing of AHRR boosted Th17-cell response to l-kynurenine, as reflected by increased levels of CYP1A1, the main gene controlled by AhR; and decreased IL17A expression. Blockade of AHRR limited the differentiation of naïve CD4-cells into Th17 lymphocytes; and modulated Th17-cell metabolic profile by increasing the levels of uridine via ATP depletion or pyrimidine salvage. Treatment with 2'-deoxy-2'-fluoro-d-arabinonucleic acid (FANA) oligonucleotides to silence human AHRR in vivo, reduced ALT levels, attenuated lymphocyte infiltration on histology, and heightened frequencies of regulatory immune subsets in NOD/scid/gamma mice, reconstituted with human CD4 cells, and exposed to Con-A. In conclusion, blockade of AHRR in AIH restores Th17-cell response to AHR, and limits Th17-cell differentiation through generation of uridine. In vivo, silencing of AHRR attenuates liver damage in NOD/scid/gamma mice. Blockade of AHRR might therefore represent a novel therapeutic strategy to modulate effector Th17-cell immunity and restore homeostasis in AIH.

CYP1A1 immunohistochemistry is highly specific for angiofibroma of soft tissue among morphological mimics.

AIMS: Angiofibroma of soft tissue (AFST) is a benign, morphologically distinctive tumour type that harbours recurrent AHRR::NCOA2 fusions in 60-70% of cases and shows a non-specific immunophenotype, expressing EMA in roughly half of cases. The AHRR::NCOA2 fusion results in increased expression of cytochrome P450 1A1 (CYP1A1); a recent study demonstrated CYP1A1 immunohistochemistry (IHC) to be moderately sensitive and highly specific for AFST. METHODS AND RESULTS: In this study, we sought to validate these findings in a larger independent cohort of 30 AFST, as well as 215 morphological mimics, including 30 solitary fibrous tumours, 29 myxoid liposarcomas, 28 low-to-intermediate grade myxofibrosarcomas (MFS), 20 atypical spindle cell lipomatous tumours (ASCLT), 20 cellular angiofibromas, 10 cases each of spindle cell lipoma, neurofibroma, malignant peripheral nerve sheath tumour, superficial angiomyxoma, cellular myxoma, soft tissue perineurioma and deep fibrous histiocytoma, and nine cases each of low-grade fibromyxoid sarcoma and mammary-type myofibroblastoma. We found CYP1A1 IHC to be 70% sensitive for AFST, with granular cytoplasmic staining in 21 of 30 tumours, and 98% specific, with staining in only five morphological mimics: two deep fibrous histiocytomas, one MFS, one cellular angiofibroma and one ASCLT. CONCLUSIONS: These findings confirm that CYP1A1 is 70% sensitive, consistent with the prevalence of AHRR::NCOA2 fusions that up-regulate this protein, and that it is highly specific among morphological mimics.

Association of the AhR, ARNT, and AhRR gene polymorphisms and cord blood AhR levels with elevated cord blood IgE susceptibility in Taiwan mother-infant pairs: a nested case-control study.

The roles of aryl hydrocarbon receptor (AhR), AhR-nuclear translocator (ARNT), and AhR repressor (AhRR) genes in the elevation of cord blood IgE (CbIgE) remained unclear. Our aims were to determine the polymorphisms of AhR, ARNT, and AhRR genes, cord blood AhR (CBAhR) level, and susceptibility to elevation of CbIgE. 206 infant-mother pairs with CbIgE>=0.35 IU/ml and 421 randomly selected controls recruited from our previous study. Genotyping was determined using TaqMan assays. Statistical analysis showed AhR rs2066853 (GG vs. AA+AG: adjusted OR (AOR)=1.5, 95%CI=1.10-2.31 and AOR=1.60, 95%CI=1.06-2.43, respectively) and the combination of AhR rs2066853 and maternal total IgE (mtIgE)>=100 IU/ml were significantly correlated with CbIgE>=0.35 IU/ml or CbIgE>=0.5 IU/ml. CBAhR in a random subsample and CbIgE levels were significantly higher in infants with rs2066853GG genotype. We suggest that infant AhR rs2066853 and their interactions with mtIgE>=100 IU/ml significantly correlate with elevated CbIgE, but AhRR and ARNT polymorphisms do not.

Clinicopathologic and genetic characterization of angiofibroma of soft tissue: a study of 12 cases including two cases with AHRR::NCOA3 gene fusion.

AIMS: Angiofibroma of soft tissue (AFST) is a benign tumour characterised by prominent arborizing blood vessels throughout the lesion. Approximately two-thirds of AFST cases were reported to have AHRR::NCOA2 fusion, and only two cases have been reported to have other gene fusions: GTF2I::NCOA2 or GAB1::ABL1. Although AFST is included in fibroblastic and myofibroblastic tumours in the World Health Organization's 2020 classification, histiocytic markers, especially CD163, have been reported to be positive in almost all examined cases, and it still remains the possibility of a fibrohistiocytic nature of the tumour. Therefore, we aimed to clarify the genetic and pathological spectrum of AFST and identify whether histiocytic marker-positive cells were true neoplastic cells. METHODS AND RESULTS: We evaluated 12 AFST cases, which included 10 cases with AHRR::NCOA2 and two with AHRR::NCOA3 fusions. Pathologically, nuclear palisading, which has not been reported in AFST, was detected in two cases. Furthermore, one tumour resected by additional wide resection revealed severe infiltrative growth. Immunohistochemical analysis indicated varying levels of desmin-positive cells in nine cases, whereas CD163- and CD68-positive cells were diffusely distributed in all 12 cases. We also performed double immunofluorescence staining and immunofluorescence in situ hybridisation in four resected cases with >10% desmin-positive tumour cells. The results suggested that the CD163-positive cells differed from desmin-positive cells with AHRR::NCOA2 fusion in all four cases. CONCLUSION: Our findings suggested that AHRR::NCOA3 could be the second most frequent fusion gene, and histiocytic marker-positive cells are not genuine neoplastic cells in AFST.

AhRR and PPP1R3C: Potential Prognostic Biomarkers for Serous Ovarian Cancer.

The lack of effective screening and successful treatment contributes to high ovarian cancer mortality, making it the second most common cause of gynecologic cancer death. Development of chemoresistance in up to 75% of patients is the cause of a poor treatment response and reduced survival. Therefore, identifying potential and effective biomarkers for its diagnosis and prognosis is a strong critical need. Copy number alterations are frequent in cancer, and relevant for molecular tumor stratification and patients' prognoses. In this study, array-CGH analysis was performed in three cell lines and derived cancer stem cells (CSCs) to identify genes potentially predictive for ovarian cancer patients' prognoses. Bioinformatic analyses of genes involved in copy number gains revealed that AhRR and PPP1R3C expression negatively correlated with ovarian cancer patients' overall and progression-free survival. These results, together with a significant association between AhRR and PPP1R3C expression and ovarian cancer stemness markers, suggested their potential role in CSCs. Furthermore, AhRR and PPP1R3C's increased expression was maintained in some CSC subpopulations, reinforcing their potential role in ovarian cancer. In conclusion, we reported for the first time, to the best of our knowledge, a prognostic role of AhRR and PPP1R3C expression in serous ovarian cancer.

The association between DNA methylation of 6p21.33 and AHRR in blood and coronary heart disease in Chinese population.

BACKGROUND: Early detection could significantly improve the prognosis of coronary heart disease (CHD). In-invitro diagnostic technique may provide a solution when sufficient biomarkers could be identified. Pertinent associations between blood-based aberrant DNA methylation and smoking, the pathogenesis of atherosclerosis, and CHD have been robustly demonstrated and replicated, but that studies in Chinese populations are rare. The blood-based methylation of aryl-hydrocarbon receptor repressor (AHRR) cg05575921 and 6p21.33 cg06126421 has been associated with cardiovascular mortality in Caucasians. Here, we aim to investigate whether the AHRR and 6p21.33 methylation in the blood is associated with CHD in the Chinese population. METHODS: In this case-control study, 180 CHD patients recruited at their first registration in our study center, and 184 controls randomly selected from the people who participated in the annual health examination were enrolled. Methylation intensities of 19 CpG sites, including AHRR cg05575921, 6p21.33 cg06126421, and their flanking CpG sites, were quantified by mass spectrometry. The association between methylation intensities and CHD was estimated by logistic regression analyses adjusted for covariant. RESULTS: Compared to the controls, lower methylation of 6p21.33_CpG_4.5/cg06126421 was independently associated with increased odds of being a CHD patient (OR per - 10% methylation = 1.42 after adjustment for age, gender, and batch effect; p = 0.032 by multiple testing corrections). No association between blood-based AHRR methylation and CHD was found. CONCLUSIONS: 6p21.33 methylation exhibits a significant association with CHD. The combination of 6p21.33 methylation and conventional risk factors might be an intermediate step towards the early detection of CHD.

The Participation of the Intrinsically Disordered Regions of the bHLH-PAS Transcription Factors in Disease Development.

The basic helix-loop-helix/Per-ARNT-SIM (bHLH-PAS) proteins are a family of transcription factors regulating expression of a wide range of genes involved in different functions, ranging from differentiation and development control by oxygen and toxins sensing to circadian clock setting. In addition to the well-preserved DNA-binding bHLH and PAS domains, bHLH-PAS proteins contain long intrinsically disordered C-terminal regions, responsible for regulation of their activity. Our aim was to analyze the potential connection between disordered regions of the bHLH-PAS transcription factors, post-transcriptional modifications and liquid-liquid phase separation, in the context of disease-associated missense mutations. Highly flexible disordered regions, enriched in short motives which are more ordered, are responsible for a wide spectrum of interactions with transcriptional co-regulators. Based on our in silico analysis and taking into account the fact that the functions of transcription factors can be modulated by posttranslational modifications and spontaneous phase separation, we assume that the locations of missense mutations inducing disease states are clearly related to sequences directly undergoing these processes or to sequences responsible for their regulation.

AHRR methylation in heavy smokers: associations with smoking, lung cancer risk, and lung cancer mortality.

BACKGROUND: A low level of methylation at cg05575921 in the aryl-hydrocarbon receptor repressor (AHRR) gene is robustly associated with smoking, and some studies have observed associations between cg05575921 methylation and increased lung cancer risk and mortality. To prospectively examine whether decreased methylation at cg05575921 may identify high risk subpopulations for lung cancer screening among heavy smokers, and mortality in cases, we evaluated associations between cg05575921 methylation and lung cancer risk and mortality, by histotype, in heavy smokers. METHODS: The ß-Carotene and Retinol Efficacy Trial (CARET) included enrollees ages 45-69 with ≥ 20 pack-year smoking histories and/or occupational asbestos exposure. A subset of CARET participants had cg05575921 methylation available from HumanMethylationEPIC assays of blood collected on average 4.3 years prior to lung cancer diagnosis in cases. Cg05575921 methylation ß-values were treated continuously for a 10% methylation decrease and as quintiles, where quintile 1 (Q1, referent) represents high methylation and Q5, low methylation. We used conditional logistic regression models to examine lung cancer risk overall and by histotype in a nested case-control study including 316 lung cancer cases (diagnosed through 2005) and 316 lung cancer-free controls matched on age (±5 years), sex, race/ethnicity, enrollment year, current/former smoking, asbestos exposure, and follow-up time. Mortality analyses included 372 lung cancer cases diagnosed between 1985 and 2013 with available methylation data. We used Cox proportional hazards models to examine mortality overall and by histotype. RESULTS: Decreased cg05575921 methylation was strongly associated with smoking, even in our population of heavy smokers. We did not observe associations between decreased pre-diagnosis cg05575921 methylation and increased lung cancer risk, overall or by histotype. We observed linear increasing trends for lung cancer-specific mortality across decreasing cg05575921 methylation quintiles for adenocarcinoma and small cell carcinoma (P-trends = 0.01 and 0.04, respectively). CONCLUSIONS: In our study of heavy smokers, decreased cg05575921 methylation was strongly associated with smoking but not increased lung cancer risk. The observed association between cg05575921 methylation and increased mortality in adenocarcinoma and small cell histotypes requires further examination. Our results do not support using decreased cg05575921 methylation as a biomarker for lung cancer screening risk stratification.

AHRR methylation predicts smoking status and smoking intensity in both saliva and blood DNA.

Many existing DNA repositories do not have robust characterizations of smoking, while for many currently ongoing studies, the advent of vaping has rendered traditional cotinine-based methods of determining smoking status unreliable. Previously, we have shown that methylation status at cg05575921 in whole blood DNA can reliably predict cigarette consumption. However, whether methylation status in saliva can be used similarly has yet to be established. Herein, we use DNA from 418 biochemically confirmed smokers or nonsmokers to compare and contrast the utility of cg05575921 in classifying and quantifying cigarette smoking. Using whole blood DNA, a model incorporating age, gender, and methylation status had a receiver operating characteristic (ROC) area under the curve (AUC) for predicting smoking status of 0.995 with a nonlinear demethylation response to smoking. Using saliva DNA, the ROC AUC for predicting smoking was 0.971 with the plot of the relationship of DNA methylation to daily cigarette consumption being very similar to that seen for whole blood DNA. The addition of information from another methylation marker designed to correct for cellular heterogeneity improved the AUC for saliva DNA to 0.981. Finally, in 31 subjects who reported quitting smoking 10 or more years previously, cg05575921 methylation was nonsignificantly different from controls. We conclude that DNA methylation status at cg05575921 in DNA from whole blood or saliva predicts smoking status and daily cigarette consumption. We suggest these epigenetic assessments for objectively ascertaining smoking status will find utility in research, clinical, and civil applications.

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 affect male 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 on World 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.

Perinatal polychlorinated biphenyls and polychlorinated dibenzofurans exposure are associated with DNA methylation changes lasting to early adulthood: Findings from Yucheng second generation.

Epigenome-wide DNA methylation has not been studied in men perinatally exposed to PCBs and dioxins. Therefore, we examined whether perinatal exposure to polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans (PCDFs) induces sustained methylation changes lasting to early adulthood. We used the Illumina HumanMethylation450 BeadChip to assess DNA methylation in whole blood among Yucheng second generation (people perinatal exposed to high PCBs and PCDFs) compared with referents. Thirty male offspring from the Yucheng cohort were randomly selected and matched with 30 male offspring from the Yucheng' neighborhood referents with similar backgrounds. Methylation differences between the Yucheng second generation and non-exposed referents were identified using a P value < 1.06 × 10-7. Differential DNA methylation with epigenome-wide statistical significance was observed for 20 CpGs mapped to 11 genes, and 19 CpGs were correlated with gestational levels of PCBs or PCDF toxic equivalency (PCDF-TEQ) with the same direction of effect. Among the 11 genes, AHRR and CYP1A1 are involved in the aryl hydrocarbon receptor signaling pathway known to mediate dioxin toxicity. MYO1G, FRMD4A, ARL4C, OLFM1, and WWC3 were previously reported to be related to carcinogenesis. This is the first study examining genome-wide DNA methylation among people perinatally exposed to high concentrations of PCBs and PCDFs. We observed novel differential methylation of several genes, indicating that modifications of DNA methylation associated with perinatal PCB and PCDF exposure may persist in exposed offspring for more than 20 years. Furthermore, involvement of several carcinogesis-related genes suggested a potential in utero epigenetic mechanisms.

A Four Marker Digital PCR Toolkit for Detecting Heavy Alcohol Consumption and the Effectiveness of Its Treatment.

Background.-Heavy alcohol consumption (HAC) is a shared concern of the forensic, medical and insurance underwriting communities. Unfortunately, there is a relative lack of clinically employable tools for detecting HAC and monitoring treatment response. Building on the results of 3 genome wide methylation studies, we have previously shown in a small group of samples that methylation sensitive digital PCR assays (MSdPCR) have the potential to accurately classify individuals with respect to HAC in a small set of individuals. Objective.-We now expand on those earlier findings using data and biomaterials from 143 participants with current HAC and 200 abstinent controls. Results.-We show that a set of 4 digital PCR assays that have a receiver operating characteristic (ROC) area under the curve (AUC) of 0.96 for detecting those with HAC. After a mean of 21 days of inpatient enforced abstinence, methylation status at one of these markers, cg04987734, began to revert to baseline values. Re-examination of methylation data from our smaller 2014 study with respect to this locus demonstrated a similarly significant reversion pattern at cg04987734 in association with treatment enforced abstinence. Conclusions.-We conclude that clinically implementable dPCR tools can sensitively detect the presence of HAC and that they show promise for monitoring alcohol treatment results. These dPCR tools could be useful to clinicians and researchers in monitoring those enrolled in substance use disorder treatment, employee wellness programs and insurance underwriting.

AHRR Methylation is a Significant Predictor of Mortality Risk in Framingham Heart Study.

Background.-The ability to predict mortality is useful to clinicians, policy makers and insurers. At the current time, prediction of future mortality is still an inexact process with some proposing that epigenetic assessments could play a role in improving prognostics. In past work, we and others have shown that DNA methylation status at cg05575921, a well-studied measure of smoking intensity, is also a predictor of mortality. However, the exact extent of that predictive capacity and its independence of other commonly measured mortality risk factors are unknown. Objective.-To determine the capacity of methylation to predict mortality. Method.-We analyzed the relationship of methylation at cg05575921 and cg04987734, a recently described quantitative marker of heavy alcohol consumption, to mortality in the Offspring Cohort of the Framingham Heart Study using proportional hazards survival analysis. Results.-In this group of participants (n = 2278) whose average age was 66 ± 9 years, we found that the inclusion of both cg05575921 and cg04987734 methylation to a base model consisting of age and sex only, or to a model containing 11 commonly used mortality risk factors, improved risk prediction. What is more, prediction accuracy for the base model plus methylation data was increased compared to the base model plus known predictors of mortality (CHD, COPD, or stroke). Conclusion.-Cg05575921, and to a smaller extent cg04987734, are strong predictors of mortality risk in older Americans and that incorporation of DNA methylation assessments to these and other loci may be useful to population scientists, actuaries and policymakers to better understand the relationship of environmental risk factors, such as smoking and drinking, to mortality.

Genome-wide mapping and analysis of aryl hydrocarbon receptor (AHR)- and aryl hydrocarbon receptor repressor (AHRR)-binding sites in human breast cancer cells.

The aryl hydrocarbon receptor (AHR) mediates the toxic actions of environmental contaminants, such as 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD), and also plays roles in vascular development, the immune response, and cell cycle regulation. The AHR repressor (AHRR) is an AHR-regulated gene and a negative regulator of AHR; however, the mechanisms of AHRR-dependent repression of AHR are unclear. In this study, we compared the genome-wide binding profiles of AHR and AHRR in MCF-7 human breast cancer cells treated for 24 h with TCDD using chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). We identified 3915 AHR- and 2811 AHRR-bound regions, of which 974 (35%) were common to both datasets. When these 24-h datasets were also compared with AHR-bound regions identified after 45 min of TCDD treatment, 67% (1884) of AHRR-bound regions overlapped with those of AHR. This analysis identified 994 unique AHRR-bound regions. AHRR-bound regions mapped closer to promoter regions when compared with AHR-bound regions. The AHRE was identified and overrepresented in AHR:AHRR-co-bound regions, AHR-only regions, and AHRR-only regions. Candidate unique AHR- and AHRR-bound regions were validated by ChIP-qPCR and their ability to regulate gene expression was confirmed by luciferase reporter gene assays. Overall, this study reveals that AHR and AHRR exhibit similar but also distinct genome-wide binding profiles, supporting the notion that AHRR is a context- and gene-specific repressor of AHR activity.

MTHFR methylation moderates the impact of smoking on DNA methylation at AHRR for African American young adults.

Smoking has been shown to have a large, reliable, and rapid effect on demethylation of AHRR, particularly at cg05575921, suggesting that methylation may be used as an index of cigarette consumption. Because the availability of methyl donors may also influence the degree of demethylation in response to smoking, factors that affect the activity of methylene tetrahydrofolate reductase (MTHFR), a key regulator of methyl group availability, may be of interest. In the current investigation, we examined the extent to which individual differences in methylation of MTHFR moderated the association between smoking and demethylation at cg05575921 as well as at other loci on AHRR associated with a main effect of smoking. Using a discovery sample (AIM, N = 293), and a confirmatory sample (SHAPE, N = 368) of young adult African Americans, degree of methylation of loci in the first exon of MTHFR was associated with amplification of the association between smoking and AHRR demethylation at cg05575921. However, genetic variation at a commonly studied MTHFR variant, C677T, did not influence cg05575921 methylation. The significant interaction between MTHFR methylation and the smoking-induced response at cg05575921 suggests a role for individual differences in methyl cycle regulation in understanding the effects of cigarette consumption on genome wide DNA methylation.

Genetically contextual effects of smoking on genome wide DNA methylation.

Smoking is the leading cause of death in the United States. It exerts its effects by increasing susceptibility to a variety of complex disorders among those who smoke, and if pregnant, to their unborn children. In prior efforts to understand the epigenetic mechanisms through which this increased vulnerability is conveyed, a number of investigators have conducted genome wide methylation analyses. Unfortunately, secondary to methodological limitations, these studies were unable to examine methylation in gene regions with significant amounts of genetic variation. Using genome wide genetic and epigenetic data from the Framingham Heart Study, we re-examined the relationship of smoking status to genome wide methylation status. When only methylation status is considered, smoking was significantly associated with differential methylation in 310 genes that map to a variety of biological process and cellular differentiation pathways. However, when SNP effects on the magnitude of smoking associated methylation changes are also considered, cis and trans-interaction effects were noted at a total of 266 and 4353 genes with no marked enrichment for any biological pathways. Furthermore, the SNP variation participating in the significant interaction effects is enriched for loci previously associated with complex medical illnesses. The enlarged scope of the methylome shown to be affected by smoking may better explicate the mediational pathways linking smoking with a myriad of smoking related complex syndromes. Additionally, these results strongly suggest that combined epigenetic and genetic data analyses may be critical for a more complete understanding of the relationship between environmental variables, such as smoking, and pathophysiological outcomes.

Comparison and combination of blood DNA methylation at smoking-associated genes and at lung cancer-related genes in prediction of lung cancer mortality.

Epigenome-wide association studies have established methylation patterns related to smoking, the major risk factor of lung cancer (LC), which are distinct from methylation profiles disclosed in LC patients. This study simultaneously investigated associations of smoking-associated and LC-related methylation markers with LC mortality. DNA methylation was determined by HM450K assay in baseline blood samples of 1,565 older adults in a population-based case-cohort study. The associations of 151 smoking-associated CpGs (smoCpGs) and 3,806 LC-related CpGs (caCpGs) with LC mortality were assessed by weighted Cox regression models, controlling for potential confounders. Multi-loci methylation scores were separately constructed based on smoCpGs and caCpGs. During a median follow-up of 13.8 years, 60 participants who had a first diagnosis of LC died from LC. The average time between sample collection and LC diagnosis was 5.8 years. Hypomethylation at 77 smoCpGs and 121 caCpGs, and hypermethylation at 4 smoCpGs and 66 caCpGs were associated with LC mortality. The associations were much stronger for smoCpGs than for caCpGs. Hazard ratios (95% CI) were 7.82 (2.91-21.00) and 2.27 (0.75-6.85), respectively, for participants in highest quartile of Score I (based on 81 smoCpGs) and Score II (based on 187 caCpGs), compared with participants in the corresponding lower three quartiles. Score I outperformed Score II, with an optimism-corrected C-index of 0.87 vs. 0.77. In conclusion, although methylation changes of both smoking-associated and LC-related genes are associated with LC mortality, only smoking-associated methylation markers predict LC mortality with high accuracy, and may thus serve as promising candidates to identify high risk populations for LC screening.

Different regulation of aryl hydrocarbon receptor-regulated genes in response to dioxin in undifferentiated and neuronally differentiated human neuroblastoma SH-SY5Y cells.

Some environmental pollutants derived from industrial processes have been suggested to be responsible for neurological impairment in children, especially in heavily polluted areas. Since these compounds are usually activators of aryl hydrocarbon receptor (AhR), it would be important to better understand the molecular pathways downstream of AhR leading to neural deficits. To this purpose, appropriate in vitro human neural model is much needed. Here we have investigated whether undifferentiated and neuronally differentiated human neuroblastoma cells, SH-SY5Y cells, can provide a suitable model for monitoring AhR activity induced by environmental pollutants, focusing on 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD), a known activator of AhR. Further characterization of differentiated SH-SY5Y showed an increase in AhRR (aryl hydrocarbon receptor repressor), no change in ARNT1 (AhR nuclear translocator 1), and a decrease in ARNT2 expression with differentiation; in contrast, AhR was undetectable in both undifferentiated and differentiated cells. Nonetheless, treatment of parental as well as differentiated SH-SY5Y cells with TCDD resulted in the induction of AhR-regulated genes, CYP1A1 and CYP1B1; AhRR expression was also affected, but to a much smaller extent. These results indicate that undifferentiated SH-SY5Y are less sensitive to TCDD than neuronally differentiated ones, suggesting a higher resistance of the undifferentiated tumor cells to toxic insults. They also suggest that TCDD in these cells may not act via direct activation of AhR that is undetectable in SH-SY5Y as well as in differentiated neurons. Hence, these cells do not provide an appropriate model for studying ligand-mediated activation of AhR.

Aromatic Hydrocarbon Receptor Repressor (AHRR) is a biomarker of ambient air pollution exposure and Coronary Artery Disease (CAD).

Two hundred and twenty subjects were recruited while undergoing cardiac catheterization. AHRR cg05575921 methylation was shown to be significantly decreased in ever smokers compared to never smokers (Mean± SD = 64.2 ± 17.2 vs 80.1 ± 11.1 respectively; P < 0.0001). In addition, higher urinary levels of 2-OHNAP and 2-OHFLU were significantly associated with more AHRR cg05575921 hypomethylation, even after correcting for smoking (ß[95%CI]= -4.161[-7.553, -0.769]; P = 0.016 and -5.190[-9.761, -0.618]; P = 0.026, respectively) but not 1-OHPYR (ß[95%CI]= -3.545 [-10.935, 3.845]; P = 0.345). Additionally, hypomethylation of AHRR ROI was significantly associated with obstructive coronary artery disease (CAD) after adjusting for smoking, age, sex, diabetes and dyslipidemia (OR [95%CI] = 1.024[1.000 - 1.048]; P = 0.046). Results of this study necessitate further validation to potentially consider clinical incorporation of AHRR methylation status as an early predictive biomarker for the potential association between ambient air pollution and CAD.