Global DNA Methylation Changes Research Articles

Global DNA Hypomethylation and Rassf1a and c-myc Promoter Hypermethylation in Rat Kidney Cells after Bisphenol A Exposure.

Bisphenol A (BPA) is a synthetic monomer used in the production of polycarbonate and an environmental contaminant with endocrine disrupting properties. BPA release from plastic carriers is thought to cause high amounts of exposure, which result in high risk to human and environment health. The study examined the possible changes in global DNA methylation, CpG promoter DNA methylation, and gene expressions of Rassf1a and c-myc after BPA exposure in rat kidney epithelial cells (NRK-52E). The IC50 values of BPA in NRK-52E cells were 133.42 and 101.74 μM in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red uptake tests, respectively. The cells were treated with BPA at 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM concentrations for 24 h and at 100 nM concentration for 24, 48, 72, 96 h, and 6 days. Decreased global 5-methylcytosine levels were observed after 48, 72, 96 h, and 6 days at the concentration of 100 nM BPA. Changes in CpG promoter DNA methylation were detected in the genes of Rassf1a and c-myc in BPA-treated NRK-52E cells. Expression levels of Rassf1a and c-myc changed in response to BPA at the high concentrations after 24 h treatment, whereas 100 nM exposure to BPA altered gene expression after 48, 72, and 96 h. These results indicate that changes in global and gene-specific DNA methylation may play an important role in the mechanism of BPA toxicity in kidney cells.

S17. GLOBAL DNA METHYLATION IN CURRENT AND EMERGENT SUICIDAL IDEATION: ANALYSIS IN SCHIZOPHRENIA

BackgroundSeveral lines of evidence have shown that epigenetic mechanisms influence suicidal behavior, but do not indicate specific susceptibility variants. In a recent study, analysis of global methylation levels found that psychiatric patients with a history of suicide attempt (SA) had significantly higher levels of 5-methyl cytosine (5mC), suggesting that patients with a history of SA have global hypermethylation of their genome. However, there is no study investigated the link between global DNA methylation and suicidal ideation (SI) in schizophrenia. In this study, we analyzed global DNA methylation in suicide ideators and non-ideators in a sample of schizophrenia patients to find if global DNA methylation can predict SI in schizophrenia.MethodsThere are several methods of detecting total 5-methylcytosine content in the genome. We used Cell Biolabs’ Global DNA Methylation ELISA Kit, which is a competitive enzyme immunoassay developed for rapid detection and quantitation of 5’-methyl-2’-deoxycytidine (5MedCyd) in DNA extracted from white blood cells (WBC). We digested the genomic DNA, extracted from WBC, into single nucleotides and the quantity of 5MedCyd was determined by comparing its absorbance with that of a known 5MedCyd standard curve. We measured global DNA methylation in predicting current and emergent suicidal ideation.ResultsIn the analysis of current SI in our schizophrenia patients, the average quantity of 5MedCyd was 7.56 ± 0.77 ng 5mC/µg DNA for suicide ideators and 8.72 ± 1.97 ng 5mC/µg DNA for non-ideators. There was no evidence of a significant difference between these two groups in our analysis (p=0.176). Also, we did not find a significant difference between global DNA methylation change (global DNA methylation change between baseline and three-month follow-up visits) in patients with and without emergent SI (p=0.121).DiscussionThis preliminary analysis did not find a predictive role of global DNA methylation in SI in patients with schizophrenia. The small sample size, used in this pilot study, is probably responsible for the fact that the results found here did not support those of previous research, showing that methylation levels are higher in patients with a history of a suicide attempt.

Recurrent Water Deficit and Epigenetic Memory in Medicago sativa L. Varieties

Global DNA methylation changes in response to recurrent drought stress were investigated in two common Greek Medicago sativa L. varieties (Lamia and Chaironia-Institute of Ιndustrial and Forage Crops). The water deficit was implemented in two phases. At the end of the first phase, which lasted for 60 days, the plants were cut at the height of 5 cm and were watered regularly for two months before being subjected to the second drought stress, which lasted for two weeks. Finally, the following groups of plants were formed: CC (controls both in phase I and phase II), CD2 (Controls in phase I, experiencing drought in phase II), and D1D2 (were subjected to drought in both phase I and phase II). At the end of phase II, samples were taken for global DNA methylation analysis with the Methylation Sensitive Amplification Polymorphism (MSAP) method, and all plants were harvested in order to measure the fresh and dry weight of roots and shoots. The variety Lamia responded better, especially the D1D2 group, compared to Chaironia in terms of root and shoot dry weight. Additionally, the shoots of Lamia had a constant water status for CD2 and D1D2 group of plants. According to DNA methylation analysis by the MSAP method, Lamia had lower total DNA methylation percentage after the second drought episode (D1D2) as compared to the plants CD2 that had experienced only one drought episode. On the other hand, the total DNA methylation percentage of Chaironia was almost the same in plants grown under recurrent drought stress conditions compared to control plants. In conclusion, the decrease of DNA methylation of Lamia stressed plants probably indicates the existence of an epigenetic mechanism that may render drought tolerance.

Stearoyl‐CoA desaturase 1 determines pancreatic β‐cell fate through regulation of DNA methylation pattern

Study objectiveStearoyl CoA desaturase 1 (SCD1) is a pivotal enzyme involved in saturated fatty acids metabolism that exhibits a protective role against lipotoxicity in pancreatic β‐cells. It has been shown that SCD1 deficiency contributes to impaired insulin secretion, β‐cell failure and development of type 2 diabetes (T2D). In the present study we investigated the involvement of SCD1 in regulation of DNA methylation and expression of transcription factors (TFs) responsible for maintenance of mature β‐cells function and identity.MethodsThe experiments were carried out ex vivo on pancreatic islets isolated from wild type (WT) and SCD1 knock‐out (SCD1 KO) mice, and in vitro on INS‐1E pancreatic β‐cell line, where lipotoxicity was induced by palmitic acid treatment. Morphological and functional characteristics of islets was evaluated by ELISA assays, immunofluorescence labeling and transmission electron microscopy techniques. DNA methylation and gene expression levels were assessed by bisulfite sequencing and real‐time quantitative PCR analyses, respectively.ResultsOur data demonstrated that pancreatic islets of SCD1 KO mice are characterized by different microarchitecture, secretory granules content and functional abnormalities manifested by diminished insulin secretion and greater proinsulin secretion in comparison to WT islets. On both ex vivo and in vitro models we observed significantly reduced expression of TFs crucial to maintenance of β‐cell identity such as Pdx1, MafA, Nkx6.1 and Neurod1. Interestingly, transcriptional repression of Pdx1 and MafA was related to excessive methylation of promoter regions of those TFs. In addition, we showed that downregulation of SCD1 activity/gene expression affects global DNA methylation pattern and changes methyl groups distribution within chromosomes in pancreatic β‐cells. DNA hypomethylation which occurred in SCD1‐deficient β‐cells was followed by decreased expression of DNA methyltransferase 1 (Dnmt1). We indicated that activation of AMP‐activated protein kinase (AMPK) axis influences post‐translational regulation of Dnmt1, thereby mediating the effect of SCD1 inhibition on global DNA methylation profile in β‐cells.ConclusionsTogether, our findings suggest that SCD1 is a gatekeeper that preserves epigenetic inheritance of DNA methylation pattern in β‐cells. Thus, SCD1 protects β‐cells from lipid‐derived loss of identity in methylation‐dependent manner through regulation of expression of TFs responsible for maintenance of β‐cell identity. Our results provide additional mechanistic insights toward understanding the role of SCD1 in T2D pathogenesis.Support or Funding InformationThis work was supported by grants from the National Science Center, Poland, grants no UMO‐2017/27/N/NZ3/01987 (AMD) and UMO‐2015/19/D/NZ4/03705 (JJ), and by The National Centre for Research and Development, grant no STRATEGMED 3/305813/2/NCBR/2017 (AD).

Life cycle exposure to propiconazole reduces fecundity by disrupting the steroidogenic pathway and altering DNA methylation in zebrafish (Danio rerio)

Propiconazole is fungicide widely used in agriculture, which may enter aquatic ecosystems and affect organisms. In this study, zebrafish (Danio rerio) were exposed to environmentally relevant levels of propiconazole throughout the life cycle, from embryo to sexually mature adults, and the effects on growth, reproduction, and offspring viability were investigated. To investigate the mechanisms of propiconazole action, the sex steroid hormones and the expression of genes related to the hypothalamus-pituitary-gonad-liver (HPGL) axis and DNA methylation were examined. Growth decreased in the parental zebrafish (F0) after exposure to propiconazole for 120 days. In males, increases in the levels of 17β-estradiol and vitellogenin were observed. The alterations in sex steroid hormones were regulated by the expression of genes involved with the HPGL axis. The decreases in fecundity and fertilization of the F0 was induced by the global DNA methylation, and then may result in the abnormal development of the F1. Therefore, propiconazole disrupted the steroidogenic pathway and caused changes in global DNA methylation that induced reproductive toxicity.

Inflammatory cytokines and organ dysfunction associate with the aberrant DNA methylome of monocytes in sepsis

BackgroundSepsis, a life-threatening organ dysfunction caused by a dysregulated systemic immune response to infection, associates with reduced responsiveness to subsequent infections. How such tolerance is acquired is not well understood but is known to involve epigenetic and transcriptional dysregulation.MethodsBead arrays were used to compare global DNA methylation changes in patients with sepsis, non-infectious systemic inflammatory response syndrome, and healthy controls. Bioinformatic analyses were performed to dissect functional reprogramming and signaling pathways related to the acquisition of these specific DNA methylation alterations. Finally, in vitro experiments using human monocytes were performed to test the induction of similar DNA methylation reprogramming.ResultsHere, we focused on DNA methylation changes associated with sepsis, given their potential role in stabilizing altered phenotypes. Tolerized monocytes from patients with sepsis display changes in their DNA methylomes with respect to those from healthy controls, affecting critical monocyte-related genes. DNA methylation profiles correlate with IL-10 and IL-6 levels, significantly increased in monocytes in sepsis, as well as with the Sequential Organ Failure Assessment score; the observed changes associate with TFs and pathways downstream to toll-like receptors and inflammatory cytokines. In fact, in vitro stimulation of toll-like receptors in monocytes results in similar gains and losses of methylation together with the acquisition of tolerance.ConclusionWe have identified a DNA methylation signature associated with sepsis that is downstream to the response of monocytes to inflammatory signals associated with the acquisition of a tolerized phenotype and organic dysfunction.

Folate deficiency promotes differentiation of vascular smooth muscle cells without affecting the methylation status of regulated genes.

Elevated serum homocysteine, an intermediate of cellular one-carbon metabolism, is an independent risk factor for cardiovascular disease (CVD). Folate deficiency increases serum homocysteine and may contribute to CVD progression. Vascular smooth muscle cells (VSMCs) regulate vascular contractility, but also contribute to repair processes in response to vascular injury. Nutritional deficiencies, like folate deficiency, are thought to impact on this phenotypic plasticity, possibly by epigenetic mechanisms. We have investigated the effect of folate deficiency on VSMCs in two cell culture systems representing early and late stages of smooth muscle cells differentiation. We find that folate deficiency promotes differentiation towards a more contractile phenotype as indicated by increased expression of respective marker genes. However, microarray analysis identified markers of striated muscle as the predominant gene expression change elicited by folate deficiency. These changes are not merely a reflection of cell cycle arrest, as foetal calf serum restriction or iron deficiency do not replicate the gene expression changes observed in response to folate deficiency. Folate deficiency only has a marginal effect on global DNA methylation. DNA methylation of CpG islands associated with genes regulated by folate deficiency remains unaffected. This supports our earlier findings in a mouse model system which also did not show any changes in global DNA methylation in response to folate and vitamin B6/B12 deficiency. These data suggest that folate deficiency enhances the expression of smooth muscle marker gene expression, promotes a shift towards a skeletal muscle phenotype, and does not regulate gene expression via DNA methylation.

Elimination of human folypolyglutamate synthetase alters programming and plasticity of somatic cells.

Folates are vital cofactors for the regeneration of S-adenosyl methionine, which is the methyl source for DNA methylation, protein methylation, and other aspects of one-carbon (C1) metabolism. Thus, folates are critical for establishing and preserving epigenetic programming. Folypolyglutamate synthetase (FPGS) is known to play a crucial role in the maintenance of intracellular folate levels. Therefore, any modulation in FPGS is expected to alter DNA methylation and numerous other metabolic pathways. To explore the role of polyglutamylation of folate, we eliminated both isoforms of FPGS in human cells (293T), producing FPGS knockout (FPGSko) cells. The elimination of FPGS significantly decreased cell proliferation, with a major effect on oxidative phosphorylation and a lesser effect on glycolysis. We found a substantial reduction in global DNA methylation and noteworthy changes in gene expression related to C1 metabolism, cell division, DNA methylation, pluripotency, Glu metabolism, neurogenesis, and cardiogenesis. The expression levels of NANOG, octamer-binding transcription factor 4, and sex-determining region Y-box 2 levels were increased in the mutant, consistent with the transition to a stem cell-like state. Gene expression and metabolite data also indicate a major change in Glu and GABA metabolism. In the appropriate medium, FPGSko cells can differentiate to produce mainly cells with characteristics of either neural stem cells or cardiomyocytes.-Srivastava, A. C., Thompson, Y. G., Singhal, J., Stellern, J., Srivastava, A., Du, J., O'Connor, T. R., Riggs, A. D. Elimination of human folypolyglutamate synthetase alters programming and plasticity of somatic cells.

No genome-wide DNA methylation changes found associated with medium-term reduced graphene oxide exposure in human lung epithelial cells

ABSTRACTThe presence of nanomaterials in our everyday life is ever increasing, and so too are concerns about the possible health consequences of exposure to them. While evidence of their biological activity is growing, there is still scant knowledge of the epigenetic mechanisms that could be at play in these processes. Moreover, the great variability in the chemical and physical structures of these compounds handicaps the study of their possible health risks. Here we have synthesized reduced graphene oxide (rGO) through the thermal exfoliation/reduction of graphite oxide, and characterized the resulting material. We have then made use of Illumina’s MethylationEPIC arrays and bisulphite pyrosequencing to analyse the genome-wide and global DNA methylation dynamics associated with the medium-term exposure of human lung epithelial cells to rGO at concentrations of 1 and 10 µg/mL. The results show no genome-wide or global DNA methylation changes associated with either condition. Our observations thus suggest that medium-term rGO exposure does not have significant effects on the DNA methylation patterns of human lung epithelial cells.

Epigenome-wide association study of depression symptomatology in elderly monozygotic twins

Depression is a severe and debilitating mental disorder diagnosed by evaluation of affective, cognitive and physical depression symptoms. Severity of these symptoms strongly impacts individual’s quality of life and is influenced by a combination of genetic and environmental factors. One of the molecular mechanisms allowing for an interplay between these factors is DNA methylation, an epigenetic modification playing a pivotal role in regulation of brain functioning across lifespan. The aim of this study was to investigate if there are DNA methylation signatures associated with depression symptomatology in order to identify molecular mechanisms contributing to pathophysiology of depression. We performed an epigenome-wide association study (EWAS) of continuous depression symptomatology score measured in a cohort of 724 monozygotic Danish twins (346 males, 378 females). Through EWAS analyses adjusted for sex, age, flow-cytometry based blood cell composition, and twin relatedness structure in the data we identified depression symptomatology score to be associated with blood DNA methylation levels in promoter regions of neuropsin (KLK8, p-value = 4.7 × 10−7) and DAZ associated protein 2 (DAZAP2, p-value = 3.13 × 10−8) genes. Other top associated probes were located in gene bodies of MAD1L1 (p-value = 5.16 × 10−6), SLC29A2 (p-value = 6.15 × 10−6) and AKT1 (p-value = 4.47 × 10−6), all genes associated before with development of depression. Additionally, the following three measures (a) DNAmAge (calculated with Horvath and Hannum epigenetic clock estimators) adjusted for chronological age, (b) difference between DNAmAge and chronological age, and (c) DNAmAge acceleration were not associated with depression symptomatology score in our cohort. In conclusion, our data suggests that depression symptomatology score is associated with DNA methylation levels of genes implicated in response to stress, depressive-like behaviors, and recurrent depression in patients, but not with global DNA methylation changes across the genome.

Contribution of DNA methylation in chronic stress-induced cardiac remodeling and arrhythmias in mice.

It is recognized that stress can induce cardiac dysfunction, but the underlying mechanisms are not well understood. The present study aimed to test the hypothesis that chronic negative stress leads to alterations in DNA methylation of certain cardiac genes, which in turn contribute to pathologic remodeling of the heart. We found that mice that were exposed to chronic restraint stress (CRS) for 4 wk exhibited cardiac remodeling toward heart failure, as characterized by ventricular chamber dilatation, wall thinning, and decreased contractility. CRS also induced cardiac arrhythmias, including intermittent sinus tachycardia and bradycardia, frequent premature ventricular contraction, and sporadic atrioventricular conduction block. Circulating levels of stress hormones were elevated, and the cardiac expression of tyrosine hydroxylase, a marker of sympathetic innervation, was increased in CRS mice. Using reduced representation bisulfite sequencing, we found that although CRS did not lead to global changes in DNA methylation in the murine heart, it nevertheless altered methylation at specific genes that are associated with the dilated cardiomyopathy (DCM) (e.g., desmin) and adrenergic signaling of cardiomyocytes (ASPC) (e.g., adrenergic receptor-α1) pathways. We conclude that CRS induces cardiac remodeling and arrhythmias, potentially through altered methylation of myocardial genes associated with the DCM and ASPC pathways.-Zhang, P., Li, T., Liu, Y.-Q., Zhang, H., Xue, S.-M., Li, G., Cheng, H.-Y.M., Cao, J.-M. Contribution of DNA methylation in chronic stress-induced cardiac remodeling and arrhythmias in mice.

Dynamic changes in DNA methylation during embryonic and postnatal development of an altricial wild bird.

DNA methylation could shape phenotypic responses to environmental cues and underlie developmental plasticity. Environmentally induced changes in DNA methylation during development can give rise to stable phenotypic traits and thus affect fitness. In the laboratory, it has been shown that the vertebrate methylome undergoes dynamic reprogramming during development, creating a critical window for environmentally induced epigenetic modifications. Studies of DNA methylation in the wild are lacking, yet are essential for understanding how genes and the environment interact to affect phenotypic development and ultimately fitness. Furthermore, our knowledge of the establishment of methylation patterns during development in birds is limited. We quantified genome‐wide DNA methylation at various stages of embryonic and postnatal development in an altricial passerine bird, the great tit Parus major. While, there was no change in global DNA methylation in embryonic tissue during the second half of embryonic development, a twofold increase in DNA methylation in blood occurred between 6 and 15 days posthatch. Though not directly comparable, DNA methylation levels were higher in the blood of nestlings compared with embryonic tissue at any stage of prenatal development. This provides the first evidence that DNA methylation undergoes global change during development in a wild bird, supporting the hypothesis that methylation mediates phenotypic development. Furthermore, the plasticity of DNA methylation demonstrated during late postnatal development, in the present study, suggests a wide window during which DNA methylation could be sensitive to environmental influences. This is particularly important for our understanding of the mechanisms by which early‐life conditions influence later‐life performance. While, we found no evidence for differences in genome‐wide methylation in relation to habitat of origin, environmental variation is likely to be an important driver of variation in methylation at specific loci.

Abstract 945: A Novel Role of Cardiac DNA Methylation as a Regulator of Fibrosis in Human Diabetic Heart Failure

Human heart failure (HF) is accompanied by changes in cardiac gene expression; however, the impact of diabetes mellitus on this transcriptional regulation remains unclear. Our laboratory studies the role of epigenetics in HF and focuses on DNA methylation, commonly accepted as a negative regulator of gene expression when present in a gene’s promoter. In the current study, we hypothesized that diabetes alters cardiac DNA methylation and the transcriptome. We performed an integrated analysis of RNA-sequencing and DNA methylation using human left ventricle biopsies from HF patients to determine the effect of diabetes on this reprogramming. Our analysis identified global changes in cardiac DNA methylation as well as mRNA expression sufficient to distinguish diabetic from non-diabetic patients. Specifically, we identified significant regulation of 1,133 genes (|1.5| fold-change, P<0.05) and 9,302 methylation sites (|5%| methylation, P<0.05). Within the 391 co-regulated genes, only 174 genes had altered promoter-associated methylation. Consistent with the accepted dogma that promoter methylation inversely regulates gene expression, we found that 119 of those 174 genes displayed this inverse relationship; of these, an overwhelming majority (102) were hypomethylated in diabetic relative to non-diabetic hearts. These findings contrast the cardiac hypermethylation we have reported for ischemic HF. Gene set enrichment of the 119 inversely-regulated genes identified numerous pathways involved in fibrosis, including extracellular matrix organization (FDR<0.05, 3.9% enriched) and collagen biosynthesis (FDR<0.05, 6.3% enriched), which suggest that DNA methylation contributes to the adverse cardiac remodeling associated with diabetic heart failure. To identify putative regulators of these transcriptional and epigenetic differences, a candidate gene approach was used to reveal induction (2.5-fold, Q<0.05) and demethylation (20.5%, P<0.05) of EGR2, a known regulator of fibrosis. Furthermore, we found induction of GADD45beta (2.6-fold, Q < 0.05) a known regulator of DNA demethylation. Taken together, these observations suggest that epigenetic mechanisms underlie an etiology-specific reprogramming of cardiac fibrosis in diabetic HF.

Global DNA methylation changes in rock pigeon (Columba livia) as a sentinel species due to polycyclic aromatic hydrocarbons exposure in Tehran (Iran) as a megacity.

Global DNA methylation, as an epigenetic modifications, can be a promising genomic marker for monitoring the contaminants and predicting their adverse health effects. The study aims to assess the effects of 16 PAH concentration on the altered DNA methylation levels in the kidney and liver of rock pigeon (Columba livia), as a sentinel species, from Tehran megacity as well as 40days benzo(a)pyrene in vitro exposure: (0.1, 2.5, 5, 7.5, and 10mgkg-1 bw). Data indicated that the total LMW-PAH (low molecular weight PAHs) group (120.22, 121.34, 103.69, and 128.79ngg-1 dw in liver, kidney, skin, and muscle, respectively) in the Tehran samples have higher levels than the other PAHs groups. In addition, the DNA methylation level had negative relation with the total amount of PAHs in liver and kidney. A comparatively higher global DNA hypomethylation (by 8.65% in liver and 3.76% in kidney) was observed in birds exposed to B(a)P. Our results lead us to suggest that DNA hypomethylation in liver and kidney associated with the B(a)P may be useful biomarker discovery (more than the amount of PAH concentration in different tissues of C. livia) in urban areas. In conclusion, based on the overall results assessed, DNA methylation changes in pigeon may show a new target pathway for evaluation of environmental health.

Higher baseline global leukocyte DNA methylation is associated with MTX non-response in early RA patients

BackgroundLow-dose methotrexate (MTX) is the first-line therapy in early rheumatoid arthritis (eRA). Up to 40% of eRA patients do not benefit from MTX therapy. MTX has been shown to inhibit one-carbon metabolism, which is involved in the donation of methyl groups. In this study, we investigate baseline global DNA methylation and changes in DNA methylation during treatment in relation to clinical non-response after 3 months of MTX treatment.MethodsTwo hundred ninety-four blood samples were collected from the Treatment in the Rotterdam Early Arthritis Cohort (tREACH, ISRCTN26791028), a multicenter, stratified single-blind clinical trial of eRA patients. Global DNA (hydroxy)methylation was quantified using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and validated with a global DNA LINE-1 methylation technique. MTX response was determined as ΔDAS28. Additionally, patients were stratified into two response groups according to the European League Against Rheumatism (EULAR) response criteria. Associations between global DNA methylation and response were examined using univariate regression models adjusted for baseline DAS28, baseline erythrocyte folate levels, and body mass index (BMI).ResultsHigher baseline global DNA methylation was associated with less decrease of DAS28 (β = 0.15, p = 0.013) and with MTX non-response (OR = 0.010, 95% CI = 0.001–0.188). This result was validated in LINE-1 elements (β = 0.22, p = 0.026). Changes in global DNA (hydroxy)methylation were not associated with MTX response over 3 months.ConclusionsThese results show that higher baseline global DNA methylation in treatment naïve eRA patients is associated with decreased clinical response after 3 months of treatment of eRA patients and can be further evaluated as a predictor for MTX therapy non-response.Trial registrationISRCTN, ISRCTN26791028, registered 23 August 2007—retrospectively registered

Interplay between proinflammatory cytokines, miRNA, and tissue lesions in Anisakis-infected Sprague-Dawley rats.

BackgroundAnisakiasis is an emerging public health problem, caused by Anisakis spp. nematode larvae. Anisakiasis presents as variable and unspecific gastrointestinal and/or allergic clinical symptoms, which accounts for the high rate of misdiagnosed cases.Methodology/Principal findingsThe aim of this study was to characterize the early cellular (6–72 h p.i.) and molecular (6 h p.i.) immune response and general underlying regulatory mechanism in Anisakis infected rats. Each Sprague-Dawley rat was infected with 10 Anisakis spp. larvae by gastric intubation. Tissues with visible lesions were processed for: i) classic histopathology (HE), immunofluorescence (CD3, iNOS, S100A8/A9), and transmission electron microscopy (TEM); ii) target genes (Il1b, Il6, Il18, Ccl3, Icam1, Mmp9) and microRNA (Rat Immunopathology MIRN-104ZF plate, Quiagen) expression analysis; and iii) global DNA methylation. Histopathology revealed that Anisakis larval migration caused moderate to extensive hemorrhages in submucosal and epimysial/perimysial connective tissue. In stomach and muscle, moderate to abundant mixed inflammatory infiltrate was present, dominated by neutrophils and macrophages, while only mild infiltration was seen in intestine. Lesions were characterized by the presence of CD3+, iNOS+, and S100A8/A9+ cells. The greatest number of iNOS+ and S100A8/A9+ cells was seen in muscle. Il6, Il1b, and Ccl3 showed particularly strong expression in stomach and visceral adipose tissues, but the order of expression differed between tissues. In total, three miRNAs were differentially expressed, two in stomach (miRNA-451 and miRNA-223) and two in intestine (miRNA-451 and miRNA-672). No changes in global DNA methylation were observed in infected tissues relative to controls.Conclusions/SignificanceAnisakis infection induces strong immune responses in infected rats with marked induction of specific proinflammatory cytokines and miRNA expression. Deciphering the functional role of these cytokines and miRNAs will help in understanding the anisakiasis pathology and controversies surrounding Anisakis infection in humans.

Non-invasive detection of DNA methylation states in carcinoma and pluripotent stem cells using Raman microspectroscopy and imaging

DNA methylation plays a critical role in the regulation of gene expression. Global DNA methylation changes occur in carcinogenesis as well as early embryonic development. However, the current methods for studying global DNA methylation levels are invasive and require sample preparation. The present study was designed to investigate the potential of Raman microspectroscopy and Raman imaging as non-invasive, marker-independent and non-destructive tools for the detection of DNA methylation in living cells. To investigate global DNA methylation changes, human colon carcinoma HCT116 cells, which were hypomorphic for DNA methyltransferase 1, therefore showing a lower global DNA methylation (DNMT1−/− cells), were compared to HCT116 wildtype cells. As a model system for early embryogenesis, murine embryonic stem cells were adapted to serum-free 2i medium, leading to a significant decrease in DNA methylation. Subsequently, 2i medium -adapted cells were compared to cells cultured in serum-containing medium. Raman microspectroscopy and imaging revealed significant differences between high- and low-methylated cell types. Higher methylated cells demonstrated higher relative intensities of Raman peaks, which can be assigned to the nucleobases and 5-methylcytosine. Principal component analysis detected distinguishable populations of high- and low-methylated samples. Based on the provided data we conclude that Raman microspectroscopy and imaging are suitable tools for the real-time, marker-independent and artefact-free investigation of the DNA methylation states in living cells.

Genomic DNA methylation signatures in different tissues after ambient air particulate matter exposure

DNA methylation (5-mc) is one of the several epigenetic markers, and is generally associated with the inhibition of gene expression. Both hyper and hypo DNA methylation are associated with the diseases. Exposure to fine particles with a diameter of 2.5 μm or less (PM2.5) is a pervasive risk factor for cardiopulmonary mortality, metabolic disorders, cognition damage, and etc.. Recent reports pointed toward that these diseases were associated with the altered DNA methylation level of some specific-gene, potentially suggesting that the DNA methylation alteration was involved in the health hazard derived from the PM2.5 exposure. In this study, we systematically investigated the global DNA methylation level of most tissues, including lung, heart, testis, thymus, spleen, epididymal fat, hippocampus, kidney, live, after short and long term PM2.5 exposure. After acute PM2.5 exposure, the global hypo-methylation in DNA was observed in lung and heart. Notably, after chronic PM2.5 exposure, level of global DNA methylation decreased in most organs which included lung, testis, thymus, spleen, epididymal fat, hippocampus and blood. The present study systematically demonstrated the global DNA methylation changes by PM2.5 exposure, and put forward a possible orientation for further exploring the effects of ambient air particles exposure on the specific organs.

UHRF genes regulate programmed interdigital tissue regression and chondrogenesis in the embryonic limb

The primordium of the limb contains a number of progenitors far superior to those necessary to form the skeletal components of this appendage. During the course of development, precursors that do not follow the skeletogenic program are removed by cell senescence and apoptosis. The formation of the digits provides the most representative example of embryonic remodeling via cell degeneration. In the hand/foot regions of the embryonic vertebrate limb (autopod), the interdigital tissue and the zones of interphalangeal joint formation undergo massive degeneration that accounts for jointed and free digit morphology. Developmental senescence and caspase-dependent apoptosis are considered responsible for these remodeling processes. Our study uncovers a new upstream level of regulation of remodeling by the epigenetic regulators Uhrf1 and Uhrf2 genes. These genes are spatially and temporally expressed in the pre-apoptotic regions. UHRF1 and UHRF2 showed a nuclear localization associated with foci of methylated cytosine. Interestingly, nuclear labeling increased in cells progressing through the stages of degeneration prior to TUNEL positivity. Functional analysis in cultured limb skeletal progenitors via the overexpression of either UHRF1 or UHRF2 inhibited chondrogenesis and induced cell senescence and apoptosis accompanied with changes in global and regional DNA methylation. Uhrfs modulated canonical cell differentiation factors, such as Sox9 and Scleraxis, promoted apoptosis via up-regulation of Bak1, and induced cell senescence, by arresting progenitors at the S phase and upregulating the expression of p21. Expression of Uhrf genes in vivo was positively modulated by FGF signaling. In the micromass culture assay Uhrf1 was down-regulated as the progenitors lost stemness and differentiated into cartilage. Together, our findings emphasize the importance of tuning the balance between cell differentiation and cell stemness as a central step in the initiation of the so-called “embryonic programmed cell death” and suggest that the structural organization of the chromatin, via epigenetic modifications, may be a precocious and critical factor in these regulatory events.

Population-Based Analysis of DNA Damage and Epigenetic Effects of 1,3-Butadiene in the Mouse.

Metabolism of 1,3-butadiene, a known human and rodent carcinogen, results in formation of reactive epoxides, a key event in its carcinogenicity. Although mice exposed to 1,3-butadiene present DNA adducts in all tested tissues, carcinogenicity is limited to liver, lung, and lymphoid tissues. Previous studies demonstrated that strain- and tissue-specific epigenetic effects in response to 1,3-butadiene exposure may influence susceptibly to DNA damage and serve as a potential mechanism of tissue-specific carcinogenicity. This study aimed to investigate interindividual variability in the effects of 1,3-butadiene using a population-based mouse model. Male mice from 20 Collaborative Cross strains were exposed to 0 or 635 ppm 1,3-butadiene by inhalation (6 h/day, 5 days/week) for 2 weeks. We evaluated DNA damage and epigenetic effects in target (lung and liver) and nontarget (kidney) tissues of 1,3-butadiene-induced carcinogenesis. DNA damage was assessed by measuring N-7-(2,3,4-trihydroxybut-1-yl)-guanine (THB-Gua) adducts. To investigate global histone modification alterations, we evaluated the trimethylation and acetylation of histones H3 and H4 across tissues. Changes in global cytosine DNA methylation were evaluated from the levels of methylation of LINE-1 and SINE B1 retrotransposons. We quantified the degree of variation across strains, deriving a chemical-specific human variability factor to address population variability in carcinogenic risk, which is largely ignored in current cancer risk assessment practice. Quantitative trait locus mapping identified four candidate genes related to chromatin remodeling whose variation was associated with interstrain susceptibility. Overall, this study uses 1,3-butadiene to demonstrate how the Collaborative Cross mouse population can be used to identify the mechanisms for and quantify the degree of interindividual variability in tissue-specific effects that are relevant to chemically induced carcinogenesis.