Publications by authors named "Sabine A S Langie"

48 Publications

Upregulation of mNEIL3 in Ogg1-null cells is a potential backup mechanism for 8-oxoG repair.

Mutagenesis 2021 Oct 13. Epub 2021 Oct 13.

School of Biosciences, The University of Birmingham, Birmingham, United Kingdom.

Reactive oxygen species formation and resultant oxidative damage to DNA are ubiquitous events in cells, the homeostasis of which can be dysregulated in a range of pathological conditions. Base excision repair is the primary repair mechanism for oxidative genomic DNA damage. One prevalent oxidized base modification, 8-oxoguanine (8-oxoG) is recognised by 8-oxoguanine glycosylase-1 (OGG1) initiating removal and repair via base excision repair (BER). Surprisingly, Ogg1 null mouse embryonic fibroblasts (mOgg1 -/- MEFs) do not accumulate 8-oxoG in the genome to the extent expected. This suggests that there are back-up repair mechanisms capable of repairing 8-oxoG in the absence of OGG1. In the current study we identified components of NER (Ercc1, Ercc4, Ercc5), BER (Lig1, Tdg, Nthl1, Mpg, Mgmt, NEIL3), MMR (Mlh1, Msh2, Msh6) and DSB (Brip1, Rad51d, Prkdc) pathways that are transcriptionally elevated in mOgg1 -/- MEFs. Interestingly, all three nucleotide excision repair genes identified: Ercc1 (2.5 ± 0.2-fold), Ercc4 (1.5 ± 0.1-fold) and Ercc5 (1.7 ± 0.2-fold) have incision activity. There was also a significant functional increase in NER activity (42.0 ± 7.9%) compared to WT MEFs. We also observed up-regulation of both Neil3 mRNA (37.9 ± 1.6-fold) and protein in mOgg1 -/- MEFs. This was associated with a 3.4 ± 0.4-fold increase in NEIL3 substrate sites in genomic DNA of cells treated with BSO, consistent with the ability of NEIL3 to remove 8-oxoG oxidation products from genomic DNA. In conclusion, we suggest that in Ogg1-null cells, upregulation of multiple DNA repair proteins including incision components of the NER pathway and Neil3 are important compensatory responses to prevent accumulation of genomic 8-oxoG.
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http://dx.doi.org/10.1093/mutage/geab038DOI Listing
October 2021

DNA damage in circulating leukocytes measured with the comet assay may predict the risk of death.

Sci Rep 2021 Aug 18;11(1):16793. Epub 2021 Aug 18.

Grupo DICOMOSA, Centro de Investigaciones Científicas Avanzadas (CICA), Departamento de Psicología, Facultad de Ciencias de La Educación, Universidade da Coruña, Campus Elviña s/n, 15071, A Coruña, Spain.

The comet assay or single cell gel electrophoresis, is the most common method used to measure strand breaks and a variety of other DNA lesions in human populations. To estimate the risk of overall mortality, mortality by cause, and cancer incidence associated to DNA damage, a cohort of 2,403 healthy individuals (25,978 person-years) screened in 16 laboratories using the comet assay between 1996 and 2016 was followed-up. Kaplan-Meier analysis indicated a worse overall survival in the medium and high tertile of DNA damage (p < 0.001). The effect of DNA damage on survival was modelled according to Cox proportional hazard regression model. The adjusted hazard ratio (HR) was 1.42 (1.06-1.90) for overall mortality, and 1.94 (1.04-3.59) for diseases of the circulatory system in subjects with the highest tertile of DNA damage. The findings of this study provide epidemiological evidence encouraging the implementation of the comet assay in preventive strategies for non-communicable diseases.
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http://dx.doi.org/10.1038/s41598-021-95976-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8373872PMC
August 2021

Different epigenetic signatures of newborn telomere length and telomere attrition rate in early life.

Aging (Albany NY) 2021 06 4;13(11):14630-14650. Epub 2021 Jun 4.

Centre for Environmental Sciences, Hasselt University, Hasselt, Belgium.

Telomere length (TL) and telomere shortening are biological indicators of aging, and epigenetic associates have been found for TL in adults. However, the role of epigenetic signatures in setting newborn TL and early life telomere dynamics is unknown. In the present study, based on 247 participating newborns from the ENVIRAGE birth cohort, whole-genome DNA methylation, profiled on the Illumina MethylationEPIC BeadChip microarray, and TL were measured in cord blood. In a follow-up visit at a mean age of 4.58 years, leukocyte TL was evaluated. We combined an epigenome-wide association study and a statistical learning method with re-sampling to select CpGs and their two-way interactions to model baseline (cord blood) TL and early-life telomere attrition rate, where distinct epigenetic signatures were identified for the two outcomes. In addition, a stronger epigenetic regulation was suggested in setting newborn TL than that of telomere dynamics in early life: 47 CpGs and 7 between-CpG interactions explained 76% of the variance in baseline TLs, while 72% of the total variance in telomere attrition rate was explained by 31 CpGs and 5 interactions. Functional enrichment analysis based on the selected CpGs in the two models revealed GLUT4 translocation and immune cell signaling pathways, respectively. These CpGs and interactions, as well as the cellular pathways, are potential novel targets of further investigation of telomere biology and aging.
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http://dx.doi.org/10.18632/aging.203117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8221291PMC
June 2021

Collection and storage of human white blood cells for analysis of DNA damage and repair activity using the comet assay in molecular epidemiology studies.

Mutagenesis 2021 07;36(3):193-212

Department of Pharmacology and Toxicology, University of Navarra, C/Irunlarrea 1, 31008 Pamplona, Spain.

DNA damage and repair activity are often assessed in blood samples from humans in different types of molecular epidemiology studies. However, it is not always feasible to analyse the samples on the day of collection without any type of storage. For instance, certain studies use repeated sampling of cells from the same subject or samples from different subjects collected at different time-points, and it is desirable to analyse all these samples in the same comet assay experiment. In addition, flawless comet assay analyses on frozen samples open up the possibility of using this technique on biobank material. In this article we discuss the use of cryopreserved peripheral blood mononuclear cells (PBMCs), buffy coat (BC) and whole blood (WB) for analysis of DNA damage and repair using the comet assay. The published literature and the authors' experiences indicate that various types of blood samples can be cryopreserved with only a minor effect on the basal level of DNA damage. There is evidence to suggest that WB and PBMCs can be cryopreserved for several years without much effect on the level of DNA damage. However, care should be taken when cryopreserving WB and BCs. It is possible to use either fresh or frozen samples of blood cells, but results from fresh and frozen cells should not be used in the same dataset. The article outlines detailed protocols for the cryopreservation of PBMCs, BCs and WB samples.
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http://dx.doi.org/10.1093/mutage/geab012DOI Listing
July 2021

An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity.

Nat Protoc 2020 12 16;15(12):3844-3878. Epub 2020 Nov 16.

Department of Pharmacology & Toxicology, School for Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University, Maastricht, the Netherlands.

This optimized protocol (including links to instruction videos) describes a comet-based in vitro DNA repair assay that is relatively simple, versatile, and inexpensive, enabling the detection of base and nucleotide excision repair activity. Protein extracts from samples are incubated with agarose-embedded substrate nucleoids ('naked' supercoiled DNA) containing specifically induced DNA lesions (e.g., resulting from oxidation, UVC radiation or benzo[a]pyrene-diol epoxide treatment). DNA incisions produced during the incubation reaction are quantified as strand breaks after electrophoresis, reflecting the extract's incision activity. The method has been applied in cell culture model systems, human biomonitoring and clinical investigations, and animal studies, using isolated blood cells and various solid tissues. Once extracts and substrates are prepared, the assay can be completed within 2 d.
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http://dx.doi.org/10.1038/s41596-020-0401-xDOI Listing
December 2020

Minimum Information for Reporting on the Comet Assay (MIRCA): recommendations for describing comet assay procedures and results.

Nat Protoc 2020 12 26;15(12):3817-3826. Epub 2020 Oct 26.

Department of Nutrition, University of Oslo, Oslo, Norway.

The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assay (MIRCA) providing recommendations for describing comet assay conditions and results. These recommendations differentiate between 'desirable' and 'essential' information: 'essential' information refers to the precise details that are necessary to assess the quality of the experimental work, whereas 'desirable' information relates to technical issues that might be encountered when repeating the experiments. Adherence to MIRCA recommendations should ensure that comet assay results can be easily interpreted and independently verified by other researchers.
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http://dx.doi.org/10.1038/s41596-020-0398-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7688437PMC
December 2020

Potassium bromate as positive assay control for the Fpg-modified comet assay.

Mutagenesis 2020 09;35(4):341-348

Department of Nutrition, University of Oslo, Sognsvannsveien 9, 0372, Oslo, Norway.

The comet assay is a popular assay in biomonitoring studies. DNA strand breaks (or unspecific DNA lesions) are measured using the standard comet assay. Oxidative stress-generated DNA lesions can be measured by employing DNA repair enzymes to recognise oxidatively damaged DNA. Unfortunately, there has been a tendency to fail to report results from assay controls (or maybe even not to employ assay controls). We believe this might have been due to uncertainty as to what really constitutes a positive control. It should go without saying that a biomonitoring study cannot have a positive control group as it is unethical to expose healthy humans to DNA damaging (and thus potentially carcinogenic) agents. However, it is possible to include assay controls in the analysis (here meant as a cryopreserved sample of cells i.e. included in each experiment as a reference sample). In the present report we tested potassium bromate (KBrO3) as a positive comet assay control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. Ten laboratories used the same procedure for treatment of monocytic THP-1 cells with KBrO3 (0.5, 1.5 and 4.5 mM for 1 h at 37°C) and subsequent cryopreservation. Results from one laboratory were excluded in the statistical analysis because of technical issues in the Fpg-modified comet assay. All other laboratories found a concentration-response relationship in cryopreserved samples (regression coefficients from 0.80 to 0.98), although with different slopes ranging from 1.25 to 11.9 Fpg-sensitive sites (%DNA in tail) per 1 mM KBrO3. Our results demonstrate that KBrO3 is a suitable positive comet assay control.
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http://dx.doi.org/10.1093/mutage/geaa011DOI Listing
September 2020

Regenerative responses following DNA damage - β-catenin mediates head regrowth in the planarian .

J Cell Sci 2020 04 24;133(8). Epub 2020 Apr 24.

Zoology, Biodiversity and Toxicology, Centre for Environmental Sciences, Hasselt University, 3590 Diepenbeek, Belgium

Pluripotent stem cells hold great potential for regenerative medicine. Increased replication and division, such is the case during regeneration, concomitantly increases the risk of adverse outcomes through the acquisition of mutations. Seeking for driving mechanisms of such outcomes, we challenged a pluripotent stem cell system during the tightly controlled regeneration process in the planarian Exposure to the genotoxic compound methyl methanesulfonate (MMS) revealed that despite a similar DNA-damaging effect along the anteroposterior axis of intact animals, responses differed between anterior and posterior fragments after amputation. Stem cell proliferation and differentiation proceeded successfully in the amputated heads, leading to regeneration of missing tissues. Stem cells in the amputated tails showed decreased proliferation and differentiation capacity. As a result, tails could not regenerate. Interference with the body-axis-associated component increased regenerative success in tail fragments by stimulating proliferation at an early time point. Our results suggest that differences in the Wnt signalling gradient along the body axis modulate stem cell responses to MMS.
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http://dx.doi.org/10.1242/jcs.237545DOI Listing
April 2020

The enzyme-modified comet assay: Enzyme incubation step in 2 vs 12-gels/slide systems.

Mutat Res Genet Toxicol Environ Mutagen 2019 Sep 22;845:402981. Epub 2018 Nov 22.

Department of Pharmacology and Toxicology, University of Navarra, C/Irunlarrea 1, 31009 Pamplona, Spain; IdiSNA, Navarra Institute for Health Research, Spain. Electronic address:

The enzyme-modified comet assay is a commonly used method to detect specific DNA lesions. However, still a lot of errors are made by many users, leading to dubious results and even misinterpretations. This technical note describes some critical points in the use of the enzyme-modified comet assay, such as the enzyme concentration, the time of incubation, the format used and the equipment. To illustrate the importance of these conditions/parameters, titration experiments of formamidopyrimidine DNA glycosylase (Fpg) were performed using the 2 gels/slide and the 12 minigels/slide formats (plus the 12-Gel Comet Assay Unit™). Incubation times of 15 and 30 min, and 1 h were used. Results showed that the 12 minigels/slide system requires a lower volume and concentration of Fpg. A longer time of incubation has a bigger impact when using such format. Moreover, the paper describes how to perform and interpret a titration experiment when using the enzyme-modified comet assay.
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http://dx.doi.org/10.1016/j.mrgentox.2018.11.005DOI Listing
September 2019

DNA repair as a human biomonitoring tool: Comet assay approaches.

Mutat Res Rev Mutat Res 2019 Jul - Sep;781:71-87. Epub 2019 Mar 6.

Department of Pharmacology & Toxicology, School for Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University, The Netherlands.

The comet assay offers the opportunity to measure both DNA damage and repair. Various comet assay based methods are available to measure DNA repair activity, but some requirements should be met for their effective use in human biomonitoring studies. These conditions include i) robustness of the assay, ii) sources of inter- and intra-individual variability must be known, iii) DNA repair kinetics should be assessed to optimize sampling timing; and iv) DNA repair in accessible surrogate tissues should reflect repair activity in target tissues prone to carcinogenic effects. DNA repair phenotyping can be performed on frozen and fresh samples, and is a more direct measurement than genomic or transcriptomic approaches. There are mixed reports concerning the regulation of DNA repair by environmental and dietary factors. In general, exposure to genotoxic agents did not change base excision repair (BER) activity, whereas some studies reported that dietary interventions affected BER activity. On the other hand, in vitro and in vivo studies indicated that nucleotide excision repair (NER) can be altered by exposure to genotoxic agents, but studies on other life style related factors, such as diet, are rare. Thus, crucial questions concerning the factors regulating DNA repair and inter-individual variation remain unanswered. Intra-individual variation over a period of days to weeks seems limited, which is favourable for DNA repair phenotyping in biomonitoring studies. Despite this reported low intra-individual variation, timing of sampling remains an issue that needs further investigation. A correlation was reported between the repair activity in easily accessible peripheral blood mononuclear cells (PBMCs) and internal organs for both NER and BER. However, no correlation was found between tumour tissue and blood cells. In conclusion, although comet assay based approaches to measure BER/NER phenotypes are feasible and promising, more work is needed to further optimize their application in human biomonitoring and intervention studies.
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http://dx.doi.org/10.1016/j.mrrev.2019.03.002DOI Listing
March 2020

The Influence of the Duration of Breastfeeding on the Infant's Metabolic Epigenome.

Nutrients 2019 Jun 22;11(6). Epub 2019 Jun 22.

Department of Public Health and Primary Care, Environment and Health, KU Leuven-University of Leuven, 3000 Leuven, Belgium.

Nutrition in the postnatal period is associated with metabolic programming. One of the presumed underlying mechanisms involves epigenetic modifications (e.g., DNA methylation). Breastfeeding has an unknown impact on DNA methylation at a young age. Within the Maternal Nutrition and Offspring's Epigenome (MANOE) study, we assessed the effect of breastfeeding duration on infant growth and buccal methylation in obesity-related genes ( = 101). A significant difference was found between infant growth and buccal and methylation at 12 months of breastfeeding. For CpG2 methylation, a positive association was found with duration of breastfeeding (slope = 0.217; 95% confidence interval (CI) 1.03, 0.330; < 0.001). For CpG3 and CpG, mean methylation levels were significantly lower when children were breastfed for 4-6 months compared to non-breastfed children (only CpG3), and those breastfed for 7-9 months, 10-12 months, or 1-3 months. On the other hand, higher CpG3 methylation was observed when mothers breastfed 7-9 months (6.1%) as compared to breastfeeding for 1-3 months (4.3%; = 0.007) and 10-12 months (4.6%; = 0.04). In addition, we observed that infant weight was significantly lower when children were breastfed for 10-12 months. Breastfeeding duration was associated with epigenetic variations in and at 12 months and with infant biometry/growth. Our results support the hypothesis that breastfeeding could induce epigenetic changes in infants.
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http://dx.doi.org/10.3390/nu11061408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6628078PMC
June 2019

Impact of Weight Loss Strategies on Obesity-Induced DNA Damage.

Mol Nutr Food Res 2019 09 14;63(17):e1900045. Epub 2019 Jun 14.

Department of Internal Medicine I, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria.

Scope: Obesity causes DNA damage, which is causally related to several disorders including cancer, infertility, and cognitive dysfunctions. The aim of this study is to investigate whether weight loss improves the integrity of the genetic material.

Methods And Results: Overweight mice are fed ad libitum either with a Western diet (WD), with a 40% caloric restricted WD, or with a high carbohydrate low protein (HCLP) diet. Caloric restriction and also the HCLP diet lead to ca. 30% weight loss, which is paralleled by decreased DNA damage ("comet" formation) and oxidative damage of purines in inner organs, additionally the activity of nucleotide excision repair increased. The effects are more pronounced in animals that have received the HCLP chow. Results of biochemical analyses indicate that the reduction of DNA damage is associated with a decrease of pro-inflammatory cytokines and lower insulin levels.

Conclusion: The study indicates that weight loss may prevent obesity-associated adverse health effects due to reduction of overall DNA damage.
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http://dx.doi.org/10.1002/mnfr.201900045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771950PMC
September 2019

Meta-analysis of epigenome-wide association studies in neonates reveals widespread differential DNA methylation associated with birthweight.

Nat Commun 2019 04 23;10(1):1893. Epub 2019 Apr 23.

Department of Biological Psychology, Netherlands Twin Register, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.

Birthweight is associated with health outcomes across the life course, DNA methylation may be an underlying mechanism. In this meta-analysis of epigenome-wide association studies of 8,825 neonates from 24 birth cohorts in the Pregnancy And Childhood Epigenetics Consortium, we find that DNA methylation in neonatal blood is associated with birthweight at 914 sites, with a difference in birthweight ranging from -183 to 178 grams per 10% increase in methylation (P < 1.06 x 10). In additional analyses in 7,278 participants, <1.3% of birthweight-associated differential methylation is also observed in childhood and adolescence, but not adulthood. Birthweight-related CpGs overlap with some Bonferroni-significant CpGs that were previously reported to be related to maternal smoking (55/914, p = 6.12 x 10) and BMI in pregnancy (3/914, p = 1.13x10), but not with those related to folate levels in pregnancy. Whether the associations that we observe are causal or explained by confounding or fetal growth influencing DNA methylation (i.e. reverse causality) requires further research.
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http://dx.doi.org/10.1038/s41467-019-09671-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478731PMC
April 2019

Toxicity Assessment of Silver Nanoparticles in Homeostatic versus Regenerating Planarians.

Nanotoxicology 2019 05 14;13(4):476-491. Epub 2019 Feb 14.

a Centre for Environmental Sciences, Zoology, Biodiversity and Toxicology , Hasselt University , Hasselt , Belgium.

Silver nanoparticles (AgNPs) belong to the most commercialized nanomaterials, used in both consumer products and medical applications. Despite its omnipresence, in-depth knowledge on the potential toxicity of nanosilver is still lacking, especially for developing organisms. Research on vertebrates is limited due to ethical concerns, and planarians are an ideal invertebrate model to study the effects of AgNPs on stem cells and developing tissues , as regeneration mimics development by triggering massive stem cell proliferation. Our results revealed a strong interference of AgNPs with tissue- and neuroregeneration which was related to an altered stem cell cycle. The presence of a PVP-coating significantly influenced toxicity outcomes, leading to elevated DNA-damage and decreased stem cell proliferation. Non-coated AgNPs had an inhibiting effect on stem cell and early progeny numbers. Overall, regenerating tissues were more sensitive to AgNP toxicity, and careful handling and appropriate decision making is needed in AgNP applications for healing and developing tissues. We emphasize on the importance of AgNP characterization, as we showed that changes in physicochemical properties influence toxicity.
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http://dx.doi.org/10.1080/17435390.2018.1553252DOI Listing
May 2019

promoter hypermethylation in saliva of children with a respiratory allergy.

Clin Epigenetics 2018 11;10:50. Epub 2018 Apr 11.

1VITO- Sustainable Health, Boeretang 200, 2400 Mol, Belgium.

Background: The prevalence of respiratory allergy in children is increasing. Epigenetic DNA methylation changes are plausible underlying molecular mechanisms.

Results: Saliva samples collected in substudies of two longitudinal birth cohorts in Belgium (FLEHS1 & FLEHS2) were used to discover and confirm DNA methylation signatures that can differentiate individuals with respiratory allergy from healthy subjects. Genome-wide analysis with Illumina Methylation 450K BeadChips revealed 23 differentially methylated gene regions (DMRs) in saliva from 11y old allergic children (N=26) vs. controls (N=20) in FLEHS1. A subset of 7 DMRs was selected for confirmation by iPLEX MassArray analysis. First, iPLEX analysis was performed in the same 46 FLEHS1 samples for analytical confirmation of the findings obtained during the discovery phase. iPLEX results correlated significantly with the 450K array data ( <0.0001) and confirmed 4 out of the 7 DMRs. Aiming for additional biological confirmation, the 7 DMRs were analyzed using iPLEX in a substudy of an independent birth cohort (FLEHS2; N=19 cases vs. 20 controls, aged 5 years). One DMR in the promoter region showed a consistent statistically significant hypermethylation in individuals with respiratory allergy across the two birth cohorts and technologies. In addition to its involvement in TGF-β signaling and T-helper differentiation, has a regulating role in lung development.

Conclusion: is considered an interesting candidate DNA methylation marker for respiratory allergy.
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http://dx.doi.org/10.1186/s13148-018-0484-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5896137PMC
February 2019

The comet assay in human biomonitoring: cryopreservation of whole blood and comparison with isolated mononuclear cells.

Mutagenesis 2018 02;33(1):41-47

Health Unit, Flemish Institute for Technological Research (VITO), Belgium.

The comet assay is often applied in human biomonitoring. Most of the time the assay is performed with isolated peripheral blood mononuclear cells (PBMC). However, using whole blood instead of isolated cells reduces processing time, and only 20 µl is sufficient for analysis. In this study, a cryopreservation protocol for human whole blood for application in the comet assay was optimised by removing excess plasma before adding freezing medium. Cryopreservation of whole blood samples (n = 30) did not increase the detected level of strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites. Although there was no significant correlation with breaks measured in fresh whole blood, strand breaks detected in frozen whole blood were significantly correlated with breaks measured in frozen PBMC (Pearson correlation r = 0.54, P < 0.01). This correlation was however not observed for FPG-sensitive sites. Since we do not yet know the full extent to which cryopreservation might influence the blood cell population, care should be taken to ensure a similar cell type and storage conditions for all samples in one study.
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http://dx.doi.org/10.1093/mutage/gex034DOI Listing
February 2018

The next three decades of the comet assay: a report of the 11th International Comet Assay Workshop.

Mutagenesis 2017 05;32(3):397-408

Environmental Risk and Health unit, Flemish Institute of Technological Research (VITO), Boeretang 200, 2400 Mol, Belgium.

The International Comet Assay Workshops are a series of scientific conferences dealing with practical and theoretical aspects of the Comet Assay (single-cell gel electrophoresis)-a simple method for detecting DNA strand breaks. The first paper describing such an assay was published over 30 years ago in 1984 by Swedish researchers O. Ostling and K. J. Johanson. Appropriately, the theme for the 2015 meeting was looking to the future: 'The Next 3 Decades of the Comet Assay'. The programme included 25 oral and 43 poster presentations depicting the latest advances in technical developments as well as applications of the comet assay in genotoxicity testing (in vitro and in vivo) and biomonitoring of both humans and the environment. Open discussion sessions based on questions from the participants allowed exchange of practical details on current comet assay protocols. This report summarises technical issues of high importance which were discussed during the sessions. We provide information on ways to improve the assay performance, by testing for cytotoxicity, by using reference samples to reduce or allow for inter-experimental variation, and by standardising quantification of the damage, including replicates and scoring enough comets to ensure statistical validity. After 30 years of experimentation with the comet assay, we are in a position to control the important experimental parameters and make the comet assay a truly reliable method with a wealth of possible applications.
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http://dx.doi.org/10.1093/mutage/gex002DOI Listing
May 2017

The Ageing Brain: Effects on DNA Repair and DNA Methylation in Mice.

Genes (Basel) 2017 Feb 17;8(2). Epub 2017 Feb 17.

Centre for Ageing and Vitality, Human Nutrition Research Centre, Institute of Cellular Medicine, Newcastle University, Campus for Ageing and Vitality, Newcastle upon Tyne NE4 5PL, UK.

Base excision repair (BER) may become less effective with ageing resulting in accumulation of DNA lesions, genome instability and altered gene expression that contribute to age-related degenerative diseases. The brain is particularly vulnerable to the accumulation of DNA lesions; hence, proper functioning of DNA repair mechanisms is important for neuronal survival. Although the mechanism of age-related decline in DNA repair capacity is unknown, growing evidence suggests that epigenetic events (e.g., DNA methylation) contribute to the ageing process and may be functionally important through the regulation of the expression of DNA repair genes. We hypothesize that epigenetic mechanisms are involved in mediating the age-related decline in BER in the brain. Brains from male mice were isolated at 3-32 months of age. Pyrosequencing analyses revealed significantly increased methylation with ageing, which correlated inversely with expression. The reduced expression correlated with enhanced expression of methyl-CpG binding protein 2 and ten-eleven translocation enzyme 2. A significant inverse correlation between methylation at CpG-site2 and expression was also observed. BER activity was significantly reduced and associated with increased 8-oxo-7,8-dihydro-2'-deoxyguanosine levels. These data indicate that and expression can be epigenetically regulated, which may mediate the effects of ageing on DNA repair in the brain.
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http://dx.doi.org/10.3390/genes8020075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333064PMC
February 2017

Maternal intake of methyl-group donors affects DNA methylation of metabolic genes in infants.

Clin Epigenetics 2017 7;9:16. Epub 2017 Feb 7.

Department of Public Health and Primary Care, Environment and Health, KU Leuven - University of Leuven, Kapucijnenvoer 35 blok D box 7001, 3000 Leuven, Belgium.

Background: Maternal nutrition during pregnancy and infant nutrition in the early postnatal period (lactation) are critically involved in the development and health of the newborn infant. The Maternal Nutrition and Offspring's Epigenome (MANOE) study was set up to assess the effect of maternal methyl-group donor intake (choline, betaine, folate, methionine) on infant DNA methylation. Maternal intake of dietary methyl-group donors was assessed using a food-frequency questionnaire (FFQ). Before and during pregnancy, we evaluated maternal methyl-group donor intake through diet and supplementation (folic acid) in relation to gene-specific ( DMR, , , ) buccal epithelial cell DNA methylation in 6 months old infants ( = 114) via pyrosequencing. In the early postnatal period, we determined the effect of maternal choline intake during lactation (in mothers who breast-fed for at least 3 months) on gene-specific buccal DNA methylation ( = 65).

Results: Maternal dietary and supplemental intake of methyl-group donors (folate, betaine, folic acid), only in the periconception period, was associated with buccal cell DNA methylation in genes related to growth ( DMR), metabolism (), and appetite control (). A negative association was found between maternal folate and folic acid intake before pregnancy and infant (slope = -1.233, 95% CI -2.342; -0.125,  = 0.0298) and DMR methylation (slope = -0.706, 95% CI -1.242; -0.107,  = 0.0101), respectively. Positive associations were observed for maternal betaine (slope = 0.875, 95% CI 0.118; 1.633,  = 0.0241) and folate (slope = 0.685, 95% CI 0.245; 1.125,  = 0.0027) intake before pregnancy and methylation. Buccal methylation in the infant was negatively associated with maternal methyl-group donor intake in the first and second trimester of pregnancy and negatively in the third trimester. We found no clear association between maternal choline intake during lactation and buccal infant DNA methylation.

Conclusions: This study suggests that maternal dietary and supplemental intake of methyl-group donors, especially in the periconception period, can influence infant's buccal DNA methylation in genes related to metabolism, growth, appetite regulation, and maintenance of DNA methylation reactions.
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http://dx.doi.org/10.1186/s13148-017-0321-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297118PMC
July 2017

RELIC: a novel dye-bias correction method for Illumina Methylation BeadChip.

BMC Genomics 2017 01 3;18(1). Epub 2017 Jan 3.

Division of Biostatistics and Bioinformatics, Department of Environmental Health, College of Medicine, University of Cincinnati, Cincinnati, OH, USA.

Background: The Illumina Infinium HumanMethylation450 BeadChip and its successor, Infinium MethylationEPIC BeadChip, have been extensively utilized in epigenome-wide association studies. Both arrays use two fluorescent dyes (Cy3-green/Cy5-red) to measure methylation level at CpG sites. However, performance difference between dyes can result in biased estimates of methylation levels.

Results: Here we describe a novel method, called REgression on Logarithm of Internal Control probes (RELIC) to correct for dye bias on whole array by utilizing the intensity values of paired internal control probes that monitor the two color channels. We evaluate the method in several datasets against other widely used dye-bias correction methods. Results on data quality improvement showed that RELIC correction statistically significantly outperforms alternative dye-bias correction methods. We incorporated the method into the R package ENmix, which is freely available from the Bioconductor website ( https://www.bioconductor.org/packages/release/bioc/html/ENmix.html ).

Conclusions: RELIC is an efficient and robust method to correct for dye-bias in Illumina Methylation BeadChip data. It outperforms other alternative methods and conveniently implemented in R package ENmix to facilitate DNA methylation studies.
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http://dx.doi.org/10.1186/s12864-016-3426-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5209853PMC
January 2017

Salivary DNA Methylation Profiling: Aspects to Consider for Biomarker Identification.

Basic Clin Pharmacol Toxicol 2017 Sep 10;121 Suppl 3:93-101. Epub 2017 Feb 10.

Environmental Risk and Health Unit, Flemish Institute for Technological Research (VITO), Mol, Belgium.

Is it not more comfortable to spit saliva in a tube than to be pricked with a needle to draw blood to analyse your health and disease risk? Many patients, study participants and (parents of) young children undoubtedly prefer non-invasive and convenient procedures. Such procedures increase compliance rates especially for longitudinal prospective studies. Saliva is an attractive biofluid providing good quality DNA to study epigenetic mechanisms underlying disease across development. In this MiniReview, we will describe the different applications of saliva in the field of epigenetics, focusing on genomewide methylation analysis. Advantages of the use of saliva and its comparability with blood will be discussed, as will the challenges in data processing and interpretation. Knowledge gaps will be identified and suggestions given on how to improve the analysis, making saliva 'the' biofluid of choice for future biomarker initiatives in many different epidemiological and public health studies.
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http://dx.doi.org/10.1111/bcpt.12721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5644718PMC
September 2017

Dietary and supplemental maternal methyl-group donor intake and cord blood DNA methylation.

Epigenetics 2017 01 10;12(1):1-10. Epub 2016 Nov 10.

a KU Leuven - University of Leuven , Department of Public Health and Primary Care , Environment and Health , Leuven , Belgium.

Maternal nutrition is critically involved in the development and health of the fetus. We evaluated maternal methyl-group donor intake through diet (methionine, betaine, choline, folate) and supplementation (folic acid) before and during pregnancy in relation to global DNA methylation and hydroxymethylation and gene specific (IGF2 DMR, DNMT1, LEP, RXRA) cord blood methylation. A total of 115 mother-infant pairs were enrolled in the MAternal Nutrition and Offspring's Epigenome (MANOE) study. The intake of methyl-group donors was assessed using a food-frequency questionnaire. LC-MS/MS and pyrosequencing were used to measure global and gene specific methylation, respectively. Dietary intake of methyl-groups before and during pregnancy was associated with changes in LEP, DNMT1, and RXRA cord blood methylation. Statistically significant higher cord blood LEP methylation was observed when mothers started folic acid supplementation more than 6 months before conception compared with 3-6 months before conception (34.6 ± 6.3% vs. 30.1 ± 3.6%, P = 0.011, LEP CpG1) or no folic acid used before conception (16.2 ± 4.4% vs. 13.9 ± 3%, P = 0.036 for LEP CpG3 and 24.5 ± 3.5% vs. 22.2 ± 3.5%, P = 0.045 for LEP mean CpG). Taking folic acid supplements during the entire pregnancy resulted in statistically significantly higher cord blood RXRA methylation as compared with stopping supplementation in the second trimester (12.3 ± 1.9% vs. 11.1 ± 2%, P = 0.008 for RXRA mean CpG). To conclude, long-term folic acid use before and during pregnancy was associated with higher LEP and RXRA cord blood methylation, respectively. To date, pregnant women are advised to take a folic acid supplement of 400 µg/day from 4 weeks before until 12 weeks of pregnancy. Our results suggest significant epigenetic modifications when taking a folic acid supplement beyond the current advice.
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http://dx.doi.org/10.1080/15592294.2016.1257450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5270634PMC
January 2017

Whole-Genome Saliva and Blood DNA Methylation Profiling in Individuals with a Respiratory Allergy.

PLoS One 2016 21;11(3):e0151109. Epub 2016 Mar 21.

Environmental Risk and Health unit, Flemish Institute for Technological Research (VITO), Mol, Belgium.

The etiology of respiratory allergies (RA) can be partly explained by DNA methylation changes caused by adverse environmental and lifestyle factors experienced early in life. Longitudinal, prospective studies can aid in the unravelment of the epigenetic mechanisms involved in the disease development. High compliance rates can be expected in these studies when data is collected using non-invasive and convenient procedures. Saliva is an attractive biofluid to analyze changes in DNA methylation patterns. We investigated in a pilot study the differential methylation in saliva of RA (n = 5) compared to healthy controls (n = 5) using the Illumina Methylation 450K BeadChip platform. We evaluated the results against the results obtained in mononuclear blood cells from the same individuals. Differences in methylation patterns from saliva and mononuclear blood cells were clearly distinguishable (PAdj<0.001 and |Δβ|>0.2), though the methylation status of about 96% of the cg-sites was comparable between peripheral blood mononuclear cells and saliva. When comparing RA cases with healthy controls, the number of differentially methylated sites (DMS) in saliva and blood were 485 and 437 (P<0.05 and |Δβ|>0.1), respectively, of which 216 were in common. The methylation levels of these sites were significantly correlated between blood and saliva. The absolute levels of methylation in blood and saliva were confirmed for 3 selected DMS in the PM20D1, STK32C, and FGFR2 genes using pyrosequencing analysis. The differential methylation could only be confirmed for DMS in PM20D1 and STK32C genes in saliva. We show that saliva can be used for genome-wide methylation analysis and that it is possible to identify DMS when comparing RA cases and healthy controls. The results were replicated in blood cells of the same individuals and confirmed by pyrosequencing analysis. This study provides proof-of-concept for the applicability of saliva-based whole-genome methylation analysis in the field of respiratory allergy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0151109PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4801358PMC
August 2016

The comet assay: past, present, and future.

Front Genet 2015 13;6:266. Epub 2015 Aug 13.

Department of Nutrition, University of Oslo Oslo, Norway.

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http://dx.doi.org/10.3389/fgene.2015.00266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534839PMC
August 2015

Causes of genome instability: the effect of low dose chemical exposures in modern society.

Carcinogenesis 2015 Jun;36 Suppl 1:S61-88

Departamento de Medicina Genomica y Toxicologia Ambiental, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de México, México CP 04510, México.

Genome instability is a prerequisite for the development of cancer. It occurs when genome maintenance systems fail to safeguard the genome's integrity, whether as a consequence of inherited defects or induced via exposure to environmental agents (chemicals, biological agents and radiation). Thus, genome instability can be defined as an enhanced tendency for the genome to acquire mutations; ranging from changes to the nucleotide sequence to chromosomal gain, rearrangements or loss. This review raises the hypothesis that in addition to known human carcinogens, exposure to low dose of other chemicals present in our modern society could contribute to carcinogenesis by indirectly affecting genome stability. The selected chemicals with their mechanisms of action proposed to indirectly contribute to genome instability are: heavy metals (DNA repair, epigenetic modification, DNA damage signaling, telomere length), acrylamide (DNA repair, chromosome segregation), bisphenol A (epigenetic modification, DNA damage signaling, mitochondrial function, chromosome segregation), benomyl (chromosome segregation), quinones (epigenetic modification) and nano-sized particles (epigenetic pathways, mitochondrial function, chromosome segregation, telomere length). The purpose of this review is to describe the crucial aspects of genome instability, to outline the ways in which environmental chemicals can affect this cancer hallmark and to identify candidate chemicals for further study. The overall aim is to make scientists aware of the increasing need to unravel the underlying mechanisms via which chemicals at low doses can induce genome instability and thus promote carcinogenesis.
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http://dx.doi.org/10.1093/carcin/bgv031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4565613PMC
June 2015

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Carcinogenesis 2015 Jun;36 Suppl 1:S254-96

Superfund Research Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27560, USA.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology.
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http://dx.doi.org/10.1093/carcin/bgv039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480130PMC
June 2015

Comet assay to measure DNA repair: approach and applications.

Front Genet 2014 25;5:288. Epub 2014 Aug 25.

Department of Nutrition, University of Oslo Oslo, Norway.

Cellular repair enzymes remove virtually all DNA damage before it is fixed; repair therefore plays a crucial role in preventing cancer. Repair studied at the level of transcription correlates poorly with enzyme activity, and so assays of phenotype are needed. In a biochemical approach, substrate nucleoids containing specific DNA lesions are incubated with cell extract; repair enzymes in the extract induce breaks at damage sites; and the breaks are measured with the comet assay. The nature of the substrate lesions defines the repair pathway to be studied. This in vitro DNA repair assay has been modified for use in animal tissues, specifically to study the effects of aging and nutritional intervention on repair. Recently, the assay was applied to different strains of Drosophila melanogaster proficient and deficient in DNA repair. Most applications of the repair assay have been in human biomonitoring. Individual DNA repair activity may be a marker of cancer susceptibility; alternatively, high repair activity may result from induction of repair enzymes by exposure to DNA-damaging agents. Studies to date have examined effects of environment, nutrition, lifestyle, and occupation, in addition to clinical investigations.
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http://dx.doi.org/10.3389/fgene.2014.00288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4142706PMC
September 2014
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