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The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.The comet assay is commonly used to assess DNA damage. This collection of consensus protocols includes adaptations for a wide range of species and sample types, assay formats and detection of different types of DNA lesions.

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., Here, members of the hCOMET COST Action program provide a consensus statement on the Minimum Information for Reporting Comet Assays (MIRCA).

Mercury (Hg) is a ubiquitous heavy metal that originates from both natural and anthropogenic sources and is transformed in the environment to its most toxicant form, methylmercury (MeHg). Recent studies suggest that MeHg exposure can alter epigenetic modifications during embryogenesis. In this study, we examined associations between prenatal MeHg exposure and levels of cord blood DNA methylation (DNAm) by meta-analysis in up to seven independent studies (n = 1462) as well as persistence of those relationships in blood from 7 to 8 year-old children (n = 794). In cord blood, we found limited evidence of differential DNAm at cg24184221 in MED31 (β = 2.28 × 10-4, p-value = 5.87 × 10-5) in relation to prenatal MeHg exposure. In child blood, we identified differential DNAm at cg15288800 (β = 0.004, p-value = 4.97 × 10-5), also located in MED31. This repeated link to MED31, a gene involved in lipid metabolism and RNA Polymerase II transcription function, may suggest a DNAm perturbation related to MeHg exposure that persists into early childhood. Further, we found evidence for association between prenatal MeHg exposure and child blood DNAm levels at two additional CpGs: cg12204245 (β = 0.002, p-value = 4.81 × 10-7) in GRK1 and cg02212000 (β = -0.001, p-value = 8.13 × 10-7) in GGH. Prenatal MeHg exposure was associated with DNAm modifications that may influence health outcomes, such as cognitive or anthropometric development, in different populations. INMA: Main funding of the epigenetic studies in INMA were grants from Instituto de Salud Carlos III (Red INMA G03/176, CB06/02/0041), Spanish Ministry of Health (FIS-PI04/1436, FIS-PI08/1151 including FEDER funds, FIS-PI11/00610, FIS-FEDER-PI06/0867, FIS-FEDER-PI03–1615, FIS-FEDER PI16/1288, FIS-FEDER PI19/1338; Miguel Servet FEDER 15/0025 and 20/0006; FIS-FSE: 17/00260), Generalitat de Catalunya-CIRIT 1999SGR 00241, Generalitat Valenciana BEST/2020/059, Fundacio ́ La Marato ́ de TV3 (090,430), EU Commission (261357-MeDALL: Mechanisms of the Development of ALLergy), and European Research Council (268479-BREATHE: Brain dEvelopment and Air polluTion ultrafine particles in school childrEn). Korean exposome study: This study was supported by the Korean Environment Industry & Technology Institute (KEITI) through “the Environmental Health Action Program”, funded by Korea Ministry of Environment (2017001360005). MoBa: The Norwegian Mother, Father and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research, NIH/NIEHS (contract no N01-ES-75558), NIH/NINDS (grant no.1 UO1 NS 047537–01 and grant no.2 UO1 NS 047537–06A1). MoBa1 and MoBa 2 are supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (Z01-ES-49019) and Norwegian Research Council/BIOBANK (grant no 221097). The work in MoBa3 was supported in part by a Postdoctoral Fellowship grant from the Ulleval Hospitals Research Council (now under Oslo University Hospital) and travel grants from the Unger-Vetlesens foundation and the Norwegian American Womens Club, all to M.C.M.K. MoBa3 epigenomics data analyses were funded by INCA/Plan Cancer-EVA-INSERM, France, and the International Childhood Cancer Cohort Consortium (I4C), and performed by the Epigenetics Group at the International Agency for Research on Cancer (IARC, Lyon, France), A.G. was supported by the grant from INCA/Plan Cancer-EVA-INSERM (France, 2015) to Z.H. and also by the IARC Postdoctoral Fellowship, partially supported by the EC FP7 Marie Curie Actions-People-Co-funding of regional, national and international programmes (COFUND). Project Viva: The Project Viva cohort is funded by NIH grants R01HL111108, R01NR013945, and R37 HD034568. RHEA: The Rhea project was financially supported by European projects, and the Greek Ministry of Health (Program of Prevention of Obesity and Neurodevelopmental Disorders in Preschool Children, in Heraklion district, Crete, Greece: 2011–2014; ‘Rhea Plus': Primary Prevention Program of Environmental Risk Factors for Reproductive Health, and Child Health: 2012–2015). The work was also supported by MICINN (MTM2015-68140-R), Centro Nacional de Genotipado-CEGEN-PRB2-ISCIII, the H2020-EU.3.1.2. - Preventing Disease Programme (grant agreement no 874583) (ATHLETE project), and from the European Union's Horizon 2020 research and innovation programme (grant Agreement number: 733206) (Early Life stressors and Lifecycle Health (LIFECYCLE)).

The general psychopathology factor (GPF) has been proposed as a way to capture variance shared between psychiatric symptoms. Despite a growing body of evidence showing both genetic and environmental influences on GPF, the biological mechanisms underlying these influences remain unclear. In the current study, we conducted epigenome-wide meta-analyses to identify both probe- and region-level associations of DNA methylation (DNAm) with school-age general psychopathology in six cohorts from the Pregnancy And Childhood Epigenetics (PACE) Consortium. DNAm was examined both at birth (cord blood; prospective analysis) and during school-age (peripheral whole blood; cross-sectional analysis) in total samples of N=2,178 and N=2,190, respectively. At school-age, we identified one probe (cg11945228) located in the Bromodomain-containing protein 2 gene (BRD2) that negatively associated with GPF (p=8.58×10−8). We also identified a significant DMR at school-age (p=1.63×10−8), implicating the SHC Adaptor Protein 4 (SHC4) gene that has been previously implicated in multiple types of psychiatric disorders in adulthood, including obsessive compulsive disorder and major depressive disorder. In contrast, no prospective associations were identified with DNAm at birth. Taken together, results of this study revealed some evidence of an association between DNAm at school-age and GPF. Future research with larger samples is needed to further assess DNAm variation associated with GPF.

Epidemiological studies mostly focus on single environmental exposures. This study aims to systematically assess associations between a wide range of prenatal and childhood environmental exposures and cognition. The study sample included data of 1298 mother-child pairs, children were 6–11 years-old, from six European birth cohorts. We measured 87 exposures during pregnancy and 122 cross-sectionally during childhood, including air pollution, built environment, meteorology, natural spaces, traffic, noise, chemicals and life styles. The measured cognitive domains were fluid intelligence (Raven's Coloured Progressive Matrices test, CPM), attention (Attention Network Test, ANT) and working memory (N-Back task). We used two statistical approaches to assess associations between exposure and child cognition: the exposome-wide association study (ExWAS) considering each exposure independently, and the deletion-substitution-addition algorithm (DSA) considering all exposures simultaneously to build a final multiexposure model. Based on this multiexposure model that included the exposure variables selected by ExWAS and DSA models, child organic food intake was associated with higher fluid intelligence (CPM) scores (beta = 1.18; 95% CI = 0.50, 1.87) and higher working memory (N-Back) scores (0.23; 0.05, 0.41), and child fast food intake (−1.25; −2.10, −0.40), house crowding (−0.39; −0.62, −0.16), and child environmental tobacco smoke (ETS) (−0.89; −1.42, −0.35), were all associated with lower CPM scores. Indoor PM2.5 exposure was associated with lower N-Back scores (−0.09; −0.16, −0.02). Additional associations in the unexpected direction were found: Higher prenatal mercury levels, maternal alcohol consumption and child higher perfluorooctane sulfonic acid (PFOS) levels were associated with better cognitive performance; and higher green exposure during pregnancy with lower cognitive performance. This first comprehensive and systematic study of many prenatal and childhood environmental risk factors suggests that unfavourable child nutrition, family crowdedness and child indoor air pollution and ETS exposures adversely and cross-sectionally associate with cognitive function. Unexpected associations were also observed and maybe due to confounding and reverse causality., Graphical abstract Prenatal and childhood exposome-wide associations with child fluid intelligence (CPM). The exposures selected by Deletion-substitution-addition algorithm are represented by a triangle. Child fluid intelligence is mainly associated with childhood exposome, fast-food (negative association, left sided) and organic food consumption (positive, right sided), house crowding (negative, left sided), and child environmental tobacco smoke (ETS, negative, left sided). Image 1, Highlights • Previous evidence for environmental risk factor associations with neurodevelopment is based on analyses of single exposures. • We systematically analysed multiple environmental exposures in relation to child neurodevelopment. • The findings describe a list of outcome-related exposures: Diet, house crowding, indoor air pollution and tobacco smoke. • The findings show methodological complexities of analysing multiple exposures and their associations with neurodevelopment.

Increased use of 1st and 2nd generation biofuels raises concerns about health effects of new emissions. We analyzed cellular and molecular lung effects in Fisher 344 rats exposed to diesel engine exhaust emissions (DEE) from a Euro 5-classified diesel engine running on B7: petrodiesel fuel containing 7% fatty acid methyl esters (FAME), or SHB20 (synthetic hydrocarbon biofuel): petrodiesel fuel containing 7% FAME and 13% hydrogenated vegetable oil. The Fisher 344 rats were exposed for 7 consecutive days (6 h/day) or 28 days (6 h/day, 5 days/week), both with and without diesel particle filter (DPF) treatment of the exhaust in whole body exposure chambers (n = 7/treatment). Histological analysis and analysis of cytokines and immune cell numbers in bronchoalveolar lavage fluid (BALF) did not reveal adverse pulmonary effects after exposure to DEE from B7 or SHB20 fuel. Significantly different gene expression levels for B7 compared to SHB20 indicate disturbed redox signaling (Cat, Hmox1), beta-adrenergic signaling (Adrb2) and xenobiotic metabolism (Cyp1a1). Exhaust filtration induced higher expression of redox genes (Cat, Gpx2) and the chemokine gene Cxcl7 compared to non-filtered exhaust. Exposure time (7 versus 28 days) also resulted in different patterns of lung gene expression. No genotoxic effects in the lungs were observed. Overall, exposure to B7 or SHB20 emissions suggests only minor effects in the lungs.

SummaryEnvironmental exposures during early life play a critical role in life-course health, yet the molecular phenotypes underlying environmental effects on health are poorly understood. In the Human Early Life Exposome (HELIX) project, a multi-centre cohort of 1,301 mother-child pairs, we associated individual exposomes consisting of >100 chemical, physical and lifestyle exposures assessed in pregnancy and childhood, with multi-omics profiles (methylome, transcriptome, metabolome and proteins) in childhood. We identified 1,170 associations, 249 in pregnancy and 921 in childhood, which revealed potential biological responses and sources of exposure. The methylome best captures the persistent influence of pregnancy exposures, including maternal smoking; while childhood exposures were associated with features from all omics layers, revealing novel signatures for indoor air quality, essential trace elements, endocrine disruptors and weather conditions. This study provides a unique resource (https://helixomics.isglobal.org/) to guide future investigation on the biological effects of the early life exposome.

Associations between per- and polyfluoroalkyl substances (PFASs) and increased blood lipids have been repeatedly observed in humans, but a causal relation has been debated. Rodent studies show reverse effects, i.e. decreased blood cholesterol and triglycerides, occurring however at PFAS serum levels at least 100-fold higher than those in humans. This paper aims to present the main issues regarding the modulation of lipid homeostasis by the two most common PFASs, PFOS and PFOA, with emphasis on the underlying mechanisms relevant for humans. Overall, the apparent contrast between human and animal data may be an artifact of dose, with different molecular pathways coming into play upon exposure to PFASs at very low versus high levels. Altogether, the interpretation of existing rodent data on PFOS/PFOA-induced lipid perturbations with respect to the human situation is complex. From a mechanistic perspective, research on human liver cells shows that PFOS/PFOA activate the PPAR alpha pathway, whereas studies on the involvement of other nuclear receptors, like PXR, are less conclusive. Other data indicate that suppression of the nuclear receptor HNF4 alpha signaling pathway, as well as perturbations of bile acid metabolism and transport might be important cellular events that require further investigation. Future studies with human-relevant test systems would help to obtain more insight into the mechanistic pathways pertinent for humans. These studies shall be designed with a careful consideration of appropriate dosing and toxicokinetics, so as to enable biologically plausible quantitative extrapolations. Such research will increase the understanding of possible perturbed lipid homeostasis related to PFOS/ PFOA exposure and the potential implications for human health.

Oxidative DNA damage in the brain has been implicated in neurodegeneration and cognitive decline. DNA glycosylases initiate base excision repair (BER), the main pathway for oxidative DNA base lesion repair. NEIL1 and NEIL3 DNA glycosylases alter cognition in mice, the role of NEIL2 remains unclear. Here, we investigate the impact of NEIL2 and its potential overlap with NEIL1 on behavior in single and double knock-out mouse models. Neil1-/-Neil2-/- mice displayed hyperactivity, reduced anxiety and improved learning. Hippocampal oxidative DNA base lesion levels were comparable between genotypes, no mutator phenotype was found. Impaired canonical repair was thus not the cause of altered behavior. Electrophysiology indicated reduced stratum oriens afferents in the hippocampal CA1 region in Neil1-/-Neil2-/-. Within CA1, NEIL1 and NEIL2 jointly regulated transcription in genes relevant for synaptic function. Thus, we postulate a cooperative function of NEIL1 and NEIL2 in genome regulation beyond canonical BER modulating memory formation and anxiety.

Oxidative DNA damage in the brain has been implicated in neurodegeneration and cognitive decline. DNA glycosylases initiate base excision repair (BER), the main pathway for oxidative DNA base lesion repair. NEIL1 and NEIL3 DNA glycosylases affect cognition in mice, while the role of NEIL2 remains unclear. Here, we investigate the impact of NEIL2 and its potential overlap with NEIL1 on behavior in knockout mouse models. Neil1−/−Neil2−/− mice display hyperactivity, reduced anxiety and improved learning. Hippocampal oxidative DNA base lesion levels are comparable between genotypes and no mutator phenotype is found. Thus, impaired canonical repair is not likely to explain the altered behavior. Electrophysiology suggests reduced axonal activation in the hippocampal CA1 region in Neil1−/−Neil2−/− mice and lack of NEIL1 and NEIL2 causes dysregulation of genes in CA1 relevant for synaptic function. We postulate a cooperative function of NEIL1 and NEIL2 in genome regulation, beyond canonical BER, modulating behavior in mice., Gunn Hildrestrand, Veslemøy Rolseth, and Nicolas Kunath et al. examine mice lacking the NEIL1 and NEIL2 DNA glycosylases involved in base excision repair. Their results suggest that loss of both NEIL1 and NEIL2 dysregulates genes relevant to synaptic function and modulates behavior in mice.

Combustion-derived particles (CDPs), in particular from traffic, are regarded as a central contributor for adverse health effects linked to air pollution. Recently, also biomass burning has been recognized as an important source for CDPs. Here, the effects of CDPs (PM10) originating from burning of pellet, charcoal and wood on key processes associated to lung carcinogenesis were explored. Human bronchial epithelial cells (HBEC3-KT) were exposed to 2.5 μg/cm2 of CDPs for 24 h and biological effects were examined in terms of cytotoxicity, inflammation, epithelial to mesenchymal transition (EMT)-related effects, DNA damage and genotoxicity. Reduced cell migration, inflammation and modulation of various PM-associated genes were observed mainly after exposure to wood and pellet. In contrast, only particles from pellet burning induced alteration in cell proliferation and DNA damage, which resulted in cell cycle alterations. Charcoal instead, appeared in general less effective in inducing pro-carcinogenic effects. These results illustrate differences in the toxicological profile due to the CDPs source. The different chemical compounds adsorbed on CDPs seemed to be central for particle properties, leading to an activation of various cellular signaling pathways involved in early steps of cancer progression.

[Abstract] The alkaline comet assay, or single cell gel electrophoresis, is one of the most popular methods for assessing DNA damage in human population. One of the open issues concerning this assay is the identification of those factors that can explain the large inter-individual and inter-laboratory variation. International collaborative initiatives such as the hCOMET project - a COST Action launched in 2016 - represent a valuable tool to meet this challenge. The aims of hCOMET were to establish reference values for the level of DNA damage in humans, to investigate the effect of host factors, lifestyle and exposure to genotoxic agents, and to compare different sources of assay variability. A database of 19,320 subjects was generated, pooling data from 105 studies run by 44 laboratories in 26 countries between 1999 and 2019. A mixed random effect log-linear model, in parallel with a classic meta-analysis, was applied to take into account the extensive heterogeneity of data, due to descriptor, specimen and protocol variability. As a result of this analysis interquartile intervals of DNA strand breaks (which includes alkali-labile sites) were reported for tail intensity, tail length, and tail moment (comet assay descriptors). A small variation by age was reported in some datasets, suggesting higher DNA damage in oldest age-classes, while no effect could be shown for sex or smoking habit, although the lack of data on heavy smokers has still to be considered. Finally, highly significant differences in DNA damage were found for most exposures investigated in specific studies. In conclusion, these data, which confirm that DNA damage measured by the comet assay is an excellent biomarker of exposure in several conditions, may contribute to improving the quality of study design and to the standardization of results of the comet assay in human populations. This article is based upon work from COST Action hCOMET CA15132, supported by COST (European Cooperation in Science and Technology www.cost.eu) - STSM fellowships for Mirta Milić (IMROH, EU 19); IMROH, Zagreb, Croatia, Institute for Medical Research and Occupational Health (IMROH), Zagreb, Croatia, and the Ministry of Science, Education and Sports of the Republic of Croatia (Grant No. 022-0222148-2125) (EU4); Cancer Plan for PestiBG; Grant number: no ENV201401(EU 8, EU9); Italian Ministry of Education, University and Research PRIN 2005, prot. 2005058197 and Cariplo Foundation (Milan, Italy), Rif. Pratica 2007-5810 and Rif. Pratica 2010.2303 (EU 18); Associazione Italiana per la Ricerca sul Cancro (AIRC) (IG 2015/17564). (EU19); European Union Integrated Projects New Generis, 6th Framework Programme, Priority 5: Food Quality and Safety; Newborns and Genotoxic Exposure Risks, FOOD-CT-2005-016320 (EU22); ACT project No. 036APy/09 and No. 005DBB/12 (EU 24); FCT-SFRH/BPD/96196/2013, SFRH/BPD/100948/2014, Portugal (EU 26); MZ 2012/8-UKBA-8; VEGA 1/0703/13, APVV 15-0063 (EU30); Xunta de Galicia (XUGA 10605B98; INCITE08PXIB106155PR; ED481B2016/190-0; Grants ED431B2019/02), Spain (EU 32); Grant 01 173034, Ministry of Education, Science and Technological Development of the Republic of Serbia (EU 42); The Centre for Industrial and Technological Development within National Strategic Consortia for Techical Research (Industrial Research diets and food with specific characteristics for elderly, SENIFOOD); University of Navarra LE/97; Physiopathology of Obesity and Nutrition (CIBER Obn); Carlos III Health Research Institute (CB12/03/30002); Ministerio de Economia y Compatitividad (‘Ramón y Cajal’ Programme, RYC-2013-14370) of the Spanish Government for personal support (EU 45); the Ministry of Education, Youth and Sports of the Czech Republic project Healthy Aging in Industrial Environment HAIE (CZ.02.1.01/0.0/0.0/16_019/0000798) which is co-financed by the European Union (European Structural and Investment funds; Operation Programme Research, Development and Education); MYES LO 1508 (EU 46); MICRODIAB Study; ClinicalTrials.org (#NCT02231736) (EU 52); The study was funded by the Italian Ministry for Education, University and Scientific Research (MIUR) - Research No. 2005-062547 (EU14, EU53); Projects financed from Serbian Ministry of Education, Science and Technological Development #11146002, #175035, #173034 (EU 54); Mehr foundation organisation, UK (EU 55); MCTI/CNPQ No. 01/2016-Universal; FAPESC No. 09/2015; MEC/MCTI/CAPES/CNPQ/FAPS/ No. 09/2014, Brazil (CSA 6); the National Nuclear Energy Agency of Indonesia (Badan Tenaga Nuklir Nasional) with contract number 080.01.06 3447.001 001.052.A (AS4); Slovak Grant Agency (APVT-21 013202, APVT-21- 017704); Ministry of Health, Slovak Republic (2005/43-SZU-21, 2006/07- SZU-02 MZ SR, 2005/42-SZU-20

The early-life exposome influences future health and accelerated biological aging has been proposed as one of the underlying biological mechanisms. We investigated the association between more than 100 exposures assessed during pregnancy and in childhood (including indoor and outdoor air pollutants, built environment, green environments, tobacco smoking, lifestyle exposures, and biomarkers of chemical pollutants), and epigenetic age acceleration in 1,173 children aged 7 years old from the Human Early-Life Exposome project. Age acceleration was calculated based on Horvath’s Skin and Blood clock using child blood DNA methylation measured by Infinium HumanMethylation450 BeadChips. We performed an exposure-wide association study between prenatal and childhood exposome and age acceleration. Maternal tobacco smoking during pregnancy was nominally associated with increased age acceleration. For childhood exposures, indoor particulate matter absorbance (PMabs) and parental smoking were nominally associated with an increase in age acceleration. Exposure to the organic pesticide dimethyl dithiophosphate and the persistent pollutant polychlorinated biphenyl-138 (inversely associated with child body mass index) were protective for age acceleration. None of the associations remained significant after multiple-testing correction. Pregnancy and childhood exposure to tobacco smoke and childhood exposure to indoor PMabs may accelerate epigenetic aging from an early age The study received funding from the European Community’s Seventh Framework Programme (FP7/2007-206) (grant agreement no 308333) (HELIX project), the H2020-EU.3.1.2. - Preventing Disease Programme (grant agreement no 874583) (ATHLETE project), and from the European Union’s Horizon 2020 research and innovation programme (grant Agreement number: 733206) (Early Life stressors and Lifecycle Health (LIFECYCLE)). BiB received funding from the Welcome Trust (WT101597MA), from the UK Medical Research Council (MRC) and Economic and Social Science Research Council (ESRC) (MR/N024397/1). INMA was supported by grants from the Instituto de Salud Carlos III, CIBERESP, and the Generalitat de Catalunya-CIRIT. KANC was funded by the grant of the Lithuanian Agency for Science Innovation and Technology (6-04-2014_31V-66). The Norwegian Mother, Father and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research. The Rhea project was financially supported by European projects (EU FP6-2003-Food-3-NewGeneris, EU FP6. STREP Hiwate, EU FP7 ENV.2007.1.2.2.2. Project No 211250 Escape, EU FP7-2008-ENV-1.2.1.4 Envirogenomarkers, EU FP7-HEALTH-2009- single stage CHICOS, EU FP7 ENV.2008.1.2.1.6. Proposal No 226285 ENRIECO, EU- FP7- HEALTH-2012 Proposal No 308333 HELIX), and the Greek Ministry of Health (Program of Prevention of obesity and neurodevelopmental disorders in preschool children, in Heraklion district, Crete, Greece: 2011-2014; “Rhea Plus”: Primary Prevention Program of Environmental Risk Factors for Reproductive Health, and Child Health: 2012-15). We acknowledge support from the Spanish Ministry of Science and Innovation through the “Centro de Excelencia Severo Ochoa 2019-2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. OR was funded by a UKRI Future Leaders Fellowship (MR/S03532X/1). MV-U and CR-A were supported by a FI fellowship from the Catalan Government (FI-DGR 2015 and #016FI_B 00272). MC received funding from Instituto Carlos III (Ministry of Economy and Competitiveness) (CD12/00563 and MS16/00128) SI

Early life stages are vulnerable to environmental hazards and present important windows of opportunity for lifelong disease prevention. This makes early life a relevant starting point for exposome studies. The Advancing Tools for Human Early Lifecourse Exposome Research and Translation (ATHLETE) project aims to develop a toolbox of exposome tools and a Europe-wide exposome cohort that will be used to systematically quantify the effects of a wide range of community- and individual-level environmental risk factors on mental, cardiometabolic, and respiratory health outcomes and associated biological pathways, longitudinally from early pregnancy through to adolescence. Exposome tool and data development include as follows: (1) a findable, accessible, interoperable, reusable (FAIR) data infrastructure for early life exposome cohort data, including 16 prospective birth cohorts in 11 European countries; (2) targeted and nontargeted approaches to measure a wide range of environmental exposures (urban, chemical, physical, behavioral, social); (3) advanced statistical and toxicological strategies to analyze complex multidimensional exposome data; (4) estimation of associations between the exposome and early organ development, health trajectories, and biological (metagenomic, metabolomic, epigenetic, aging, and stress) pathways; (5) intervention strategies to improve early life urban and chemical exposomes, co-produced with local communities; and (6) child health impacts and associated costs related to the exposome. Data, tools, and results will be assembled in an openly accessible toolbox, which will provide great opportunities for researchers, policymakers, and other stakeholders, beyond the duration of the project. ATHLETE's results will help to better understand and prevent health damage from environmental exposures and their mixtures from the earliest parts of the life course onward. ISGlobal acknowledges support from the Spanish Ministry of Science, Innovation and Universities through the “Centro de Excelencia Severo Ochoa 2019–2023” Program (CEX2018-000806-S) and support from the Generalitat de Catalunya through the Centres de Recerca de Catalunya Program. We acknowledge collaboration with European projects, specifically: Human Early Life Exposome (HELIX) (FP7 grant agreement number 308333), LifeCycle (H2020 grant agreement number 733206), and Connecting Europe and Canada in personalized health (EUCAN-Connect) (H2020 Grant Agreement number 824989). The Born in Bradford (BiB) cohort is only possible because of the enthusiasm and commitment of the Children and Parents in BiB. We are grateful to all the participants, health professionals, and researchers who have made BiB happen. The BiB cohort is only possible because of the enthusiasm and commitment of the children and parents in BiB. We are grateful to all the participants, health professionals, schools, and researchers who have made BiB happen. BiB has received funding from the Wellcome Trust (101597), a joint grant from the UK Medical Research Council and UK Economic and Social Science Research Council (MR/N024391/1), a British Heart Foundation Clinical Study grant (CS/16/4/32482). The Norwegian Mother, Father and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research. We are grateful to all the participating families in Norway who take part in this ongoing cohort study. The Danish National Birth Cohort was established with a significant grant from the Danish National Research Foundation. Additional support was obtained from the Danish Regional Committees, the Pharmacy Foundation, the Egmont Foundation, the March of Dimes Birth Defects Foundation, the Health Foundation, and other minor grants. The Danish National Birth Cohort Biobank has been supported by the Novo Nordisk Foundation and the Lundbeck Foundation. Follow-up of mothers and children have been supported by the Danish Medical Research Council (SSVF 0646, 271-08-0839/06-066023, O602-01042B, 0602-02738B); the Lundbeck Foundation (195/04, R100-A9193); The Innovation Fund Denmark 0603-00294B (09-067124); the Nordea Foundation (02-2013-2014); Aarhus Ideas (AU R9-A959-13-S804); University of Copenhagen Strategic Grant (IFSV 2012); and the Danish Council for Independent Research (DFF—4183-00594 and DFF—4183-00152). The general design of the Generation R Study is made possible by financial support from the Erasmus MC, University Medical Center, Rotterdam, Erasmus University Rotterdam, Netherlands Organization for Health Research and Development (ZonMw), Netherlands Organisation for Scientific Research (NWO), Ministry of Health, Welfare and Sport, and Ministry of Youth and Families. This project received funding from the European Union’s Horizon 2020 research and innovation programme (LIFECYCLE, grant agreement number 733206, 2016; EUCAN-Connect grant agreement number 824989; ATHLETE, grant agreement number 874583). We gratefully acknowledge the contribution of participants, research collaborators, general practitioners, hospitals, midwives, and pharmacies in Rotterdam. The INfancia y Medio Ambiente cohort (INMA) Sababell study was funded by grants from Instituto de Salud Carlos III (Red INMA G03/176; CB06/02/0041; PI041436; PI081151 incl. Fondo Europeo de Desarollo Regional (FEDER) funds; PI12/01890 incl. FEDER funds; CP13/00054 incl. FEDER funds; PI15/00118 incl. FEDER funds; CP16/00128 incl. FEDER funds; PI16/00118 incl. FEDER funds; PI16/00261 incl. FEDER funds; PI17/01340 incl. FEDER funds, PI18/00547 incl. FEDER funds, PI20/01695 incl. FEDER funds), Centro de Investigación Biomédica en Red - Epidemiología y Salud Pública (CIBERESP), Generalitat de Catalunya-Comissió Interdepartamental de Recerca i Innovació Tecnològia 1999SGR 00241, Generalitat de Catalunya-Agencia de Gestión de Ayudas Universitarias y de Investigación (2009 SGR 501, 2014 SGR 822), Fundació La Marató de TV3 (090430), Spanish Ministry of Economy and Competitiveness (SAF2012-32991 incl. FEDER funds), Agence Nationale de Securite Sanitaire de l’Alimentation de l’Environnement et du Travail (1262C0010; EST-2016 RF-21, EST-19 RF-04), and the European Commission (261357, 308333, 603794 and 634453). The INMA Valencia study was supported by grants from Instituto de Salud Carlos III (FIS-FEDER: 13/2032, 13/1944, 14/00891, 16/1288, 17/00663, and 19/1338; Miguel Servet-FSE: CP15/0025 and MSII16/00051, 00051, and MSII20/0006), Alicia Koplowitz Foundation 2017, Generalitat Valenciana (AICO/2020/285), and Spanish Association Against Cancer (AECC) “Seed Ideas” 2019 (IDEAS19098LOPE). INMA Gipuzkoa was funded by grants from Instituto de Salud Carlos III (FIS-PI06/0867, FIS-PI09/00090, FIS-PI13/02187, and FIS-PI18/01142 incl. FEDER funds), CIBERESP, Department of Health of the Basque Government (2005111093, 2009111069, 2013111089, and 2015111065), and the Provincial Government of Gipuzkoa (DFG06/002, DFG08/001, and DFG15/221) and annual agreements with the municipalities of the study area (Zumarraga, Urretxu, Legazpi, Azkoitia y Azpeitia y Beasain). The Sepages cohort would like to thank the Grenoble University Hospital (CHU-GA) biobank (bb-0033-00069). We thank the Sepages Study group (E. Eyriey, P. Hoffmann, E. Hullo, J. Lepeule, C. Llerena, S. Lyon-Caen, X. Morin, A. Morlot, C. Philippat, I. Pin, J. Quentin, V. Siroux, R. Slama) and the participants of the Suivi de l’Exposition à la Pollution Atmosphérique durant la Grossesse et Effets sur la Santé (SEPAGES) study. We acknowledge support from Auvergne-Rhône-Alpes Région, Soutien aux coopérations universitaires et scientifiques internationales fund to support collaborations between Catalunya and Auvergne-Rhône-Alpes. SEPAGES cohort was supported by the European Research Council (consolidator grant N 311765-E-DOHaD, Principal Investigator [PI], R. Slama), by French National Research Agency (ANR), the ANR (Pregnancy, Air Pollution, Epigenetics and Respiratory health project ANR-12-PDOC-0029-01, PI, J. Lepeule; A Longitudinal Analysis of Effects of Early Life Exposure to Phenols on Health in Humans project, 14-CE21-0007-01, PI, R. Slama; Gut Microbiota in early childhood and Maternal Environmental exposures project, PI, R. Slama; Prenatal Exposure to Tobacco smoking and Air Pollution and Effects on offspring respiratory and neurodevelopmental outcomes ANR 18-CE36-005, PI, J. Lepeule). The Central European Longitudinal Study of Pregnacy and Childhood: The Next Generation (TNG) cohort is supported by the RECETOX research infrastructure (the Ministry of Education, Youth and Sports of the Czech Republic: LM2018121) and the CETOCOEN EXCELLENCE Teaming 2 project of European Union (EU) Horizon 2020 (857560) and the MEYS of the Czech Republic (02.1.01/0.0/0.0/18_ 046/0015975). The Barcelona Life Study Cohort has received funding from European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme under grant agreement number (785994) (Prenatal exposure to urban air pollution and pre and post-natal brain development project) and from the Health Effects Institute (HEI) under Agreement number 4959-RFPA15-1/18-1 (FRONTIER project). Genotyping in the HELIX study was supported by project PI17/01225 (Instituto de Salud Carlos III, co-funded by European Union [European Regional Development Fund], “A way to make Europe”) and the Centro Nacional de Genotipado-CEGEN (PRB2-ISCIII). The Nascita e Infanzia: gli Effetti dell’Ambiente cohort was partially funded by the Compagnia SanPaolo Foundation and the Piedmont Region. The Piccolipiù project was funded by the Italian National Center for Disease Prevention and Control (National Centre for Disease Prevention and Control grants years 2010 and 2014) and by the Italian Ministry of Health (art 12 and 12 bis D.lgs 502/92). The Perturbateurs Endocriniens: Étude Longitudinale sur les Anomalies de la Grossesse, l’Infertilité et l’Enfance study is supported by National Institute of Health and Medical Research and has received multiple funds from the ANR, the French Agency for Food, Environmental and Occupational Health and Safety (ANSES), the Fondation de France, the National Institute for Public Health Surveillance (InVS), the French, Ministry of Labor, the French Ministry of Health, and the French Ministry of Ecology. DataSHIELD is funded under a group of projects that underpin a program of development and application of secure methods for co-analysis, data sharing, and visualization in the Population Health Sciences Institute at Newcastle University (United Kingdom). These include: the “Connected Health Cities project” (North East and North Cumbria) funded by the UK Department of Health (RES/0150/7943/202); the “EUCanConnect project” (European Commission H2020 Flagship Collaboration with Canada); the “58FORWARDS project” (Fostering new Opportunities for Researchers via Wider Access to Research Data and Samples) funded jointly by the Wellcome Trust and the Medical Research Council (108439/Z/15/Z); and the “METADAC project” (Managing Ethico-social, Technical and Administrative issues in Data ACcess) funded jointly by the Medical Research Council, the Wellcome Trust, and the Economic and Social Research Council (MR/N01104X/1 and MR/N01104X/2). We acknowledge the Molecular Genetics Information System team, including Fleur Kelpin, Tommy de Boer, Mariska Slofstra, Connor Stroomberg, Jelmer Veen, Jeroen van Veen, Fernanda de Andrade, Marije van der Geest, Dieuwke Roelofs-Prins, Dennis Hendriksen, Bart Charbon, Joeri van der Velde, Max Postema, Erik Schaberg, Christiaan Hilbrands, Alexander Kellmann, and Luuk Dijkhuis.

Funder: Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada); doi: https://doi.org/10.13039/501100000024, Attention-deficit and hyperactivity disorder (ADHD) is a common childhood disorder with a substantial genetic component. However, the extent to which epigenetic mechanisms play a role in the etiology of the disorder is unknown. We performed epigenome-wide association studies (EWAS) within the Pregnancy And Childhood Epigenetics (PACE) Consortium to identify DNA methylation sites associated with ADHD symptoms at two methylation assessment periods: birth and school age. We examined associations of both DNA methylation in cord blood with repeatedly assessed ADHD symptoms (age 4-15 years) in 2477 children from 5 cohorts and of DNA methylation at school age with concurrent ADHD symptoms (age 7-11 years) in 2374 children from 9 cohorts, with 3 cohorts participating at both timepoints. CpGs identified with nominal significance (p < 0.05) in either of the EWAS were correlated between timepoints (ρ = 0.30), suggesting overlap in associations; however, top signals were very different. At birth, we identified nine CpGs that predicted later ADHD symptoms (p < 1 × 10-7), including ERC2 and CREB5. Peripheral blood DNA methylation at one of these CpGs (cg01271805 in the promoter region of ERC2, which regulates neurotransmitter release) was previously associated with brain methylation. Another (cg25520701) lies within the gene body of CREB5, which previously was associated with neurite outgrowth and an ADHD diagnosis. In contrast, at school age, no CpGs were associated with ADHD with p < 1 × 10-7. In conclusion, we found evidence in this study that DNA methylation at birth is associated with ADHD. Future studies are needed to confirm the utility of methylation variation as biomarker and its involvement in causal pathways.

The identification of expression quantitative trait methylation (eQTMs), defined as associations between DNA methylation levels and gene expression, might help the biological interpretation of epigenome-wide association studies (EWAS). We aimed to identify autosomal cis eQTMs in children's blood, using data from 832 children of the Human Early Life Exposome (HELIX) project.Blood DNA methylation and gene expression were measured with the Illumina 450K and the Affymetrix HTA v2 arrays, respectively. The relationship between methylation levels and expression of nearby genes (1 Mb window centered at the transcription start site, TSS) was assessed by fitting 13.6 M linear regressions adjusting for sex, age, cohort, and blood cell composition.We identified 39,749 blood autosomal cis eQTMs, representing 21,966 unique CpGs (eCpGs, 5.7% of total CpGs) and 8,886 unique transcript clusters (eGenes, 15.3% of total transcript clusters, equivalent to genes). In 87.9% of these cis eQTMs, the eCpG was located at250 kb from eGene's TSS; and 58.8% of all eQTMs showed an inverse relationship between the methylation and expression levels. Only around half of the autosomal cis-eQTMs eGenes could be captured through annotation of the eCpG to the closest gene. eCpGs had less measurement error and were enriched for active blood regulatory regions and for CpGs reported to be associated with environmental exposures or phenotypic traits. In 40.4% of the eQTMs, the CpG and the eGene were both associated with at least one genetic variant. The overlap of autosomal cis eQTMs in children's blood with those described in adults was small (13.8%), and age-shared cis eQTMs tended to be proximal to the TSS and enriched for genetic variants.This catalogue of autosomal cis eQTMs in children's blood can help the biological interpretation of EWAS findings and is publicly available at https://helixomics.isglobal.org/ and at Dryad (doi:10.5061/dryad.fxpnvx0t0).The study has received funding from the European Community's Seventh Framework Programme (FP7/2007-206) under grant agreement no 308333 (HELIX project); the H2020-EU.3.1.2. - Preventing Disease Programme under grant agreement no 874583 (ATHLETE project); from the European Union's Horizon 2020 research and innovation programme under grant agreement no 733206 (LIFECYCLE project), and from the European Joint Programming Initiative "A Healthy Diet for a Healthy Life" (JPI HDHL and Instituto de Salud Carlos III) under the grant agreement no AC18/00006 (NutriPROGRAM project). The genotyping was supported by the projects PI17/01225 and PI17/01935, funded by the Instituto de Salud Carlos III and co-funded by European Union (ERDF, "A way to make Europe") and the Centro Nacional de Genotipado-CEGEN (PRB2-ISCIII). BiB received core infrastructure funding from the Wellcome Trust (WT101597MA) and a joint grant from the UK Medical Research Council (MRC) and Economic and Social Science Research Council (ESRC) (MR/N024397/1). INMA data collections were supported by grants from the Instituto de Salud Carlos III, CIBERESP, and the Generalitat de Catalunya-CIRIT. KANC was funded by the grant of the Lithuanian Agency for Science Innovation and Technology (6-04-2014_31V-66). The Norwegian Mother, Father and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research. The Rhea project was financially supported by European projects (EU FP6-2003-Food-3-NewGeneris, EU FP6. STREP Hiwate, EU FP7 ENV.2007.1.2.2.2. Project No 211250 Escape, EU FP7-2008-ENV-1.2.1.4 Envirogenomarkers, EU FP7-HEALTH-2009- single stage CHICOS, EU FP7 ENV.2008.1.2.1.6. Proposal No 226285 ENRIECO, EU- FP7- HEALTH-2012 Proposal No 308333 HELIX), and the Greek Ministry of Health (Program of Prevention of obesity and neurodevelopmental disorders in preschool children, in Heraklion district, Crete, Greece: 2011-2014; "Rhea Plus": Primary Prevention Program of Environmental Risk Factors for Reproductive Health, and Child Health: 2012-15). We acknowledge support from the Spanish Ministry of Science and Innovation through the "Centro de Excelencia Severo Ochoa 2019-2023" Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. MV-U and CR-A were supported by a FI fellowship from the Catalan Government (FI-DGR 2015 and #016FI_B 00272). MC received funding from Instituto Carlos III (Ministry of Economy and Competitiveness) (CD12/00563 and MS16/00128).Cells can fine-tune which genes they activate, when and at which levels using a range of chemical marks on the DNA and certain proteins that help to organise the genome. One well-known example of such ‘epigenetic tags’ is DNA methylation, whereby a methyl group is added onto particular positions in the genome. Many factors – including environmental effects such as diet – control DNA methylation, allowing an organism to adapt to ever-changing conditions. An expression quantitative trait methylation (eQTM) is a specific position of the genome whose DNA methylation status regulates the activity of a given gene. A catalogue of eQTMs would be useful in helping to reveal how the environment and disease impacts the way cells work. Yet, currently, the relationships between most epigenetic tags and gene activity remains unclear, especially in children. To fill this gap, Ruiz-Arenas et al. studied DNA methylation in blood samples from over 800 healthy children across Europe. Amongst all tested DNA methylation sites, 22,000 (5.7% of total) were associated with the expression of a gene – and therefore were eQTMs; reciprocally, 9,000 genes (15.3% of all tested genes) were linked to at least one methylation site, leading to a total of 40,000 pairs of DNA methylation sites and genes. Most often, eQTMs regulated the expression of nearby genes – but only half controlled the gene that was the closest to them. Age and the genetic background of the individuals influenced the nature of eQTMs. This catalogue is a useful resource for the scientific community to start understanding the relationship between epigenetics and gene activity. Similar studies are now needed for other tissues and age ranges. Overall, extending our knowledge of eQTMs may help reveal how life events lead to illness, and could inform prevention efforts.

DNA methylation profiles of aggressive behavior may capture lifetime cumulative effects of genetic, stochastic, and environmental influences associated with aggression. Here, we report the first large meta-analysis of epigenome-wide association studies (EWAS) of aggressive behavior (N=15,324 participants). In peripheral blood samples of 14,434 participants from 18 cohorts with mean ages ranging from 7 to 68 years, 13 methylation sites were significantly associated with aggression (alpha=1.2×10−7; Bonferroni correction). In cord blood samples of 2,425 children from five cohorts with aggression assessed at mean ages ranging from 4 to 7 years, 83% of these sites showed the same direction of association with childhood aggression (r=0.74, p=0.006) but no epigenome-wide significant sites were found. Top-sites (48 at a false discovery rate of 5% in the peripherl blood meta-analysis or in a combined meta-analysis of peripheral blood and cord blood) have been associated with chemical exposures, smoking, cognition, metabolic traits, and genetic variation (mQTLs). Three genes whose expression levels were associated with top-sites were previously linked to schizophrenia and general risk tolerance. At six CpGs, DNA methylation variation in blood mirrors variation in the brain. On average 44% (range=3-82%) of the aggression–methylation association was explained by current and former smoking and BMI. These findings point at loci that are sensitive to chemical exposures with potential implications for neuronal functions. We hope these results to be a starting point for studies leading to applications as peripheral biomarkers and to reveal causal relationships with aggression and related traits.

Background: The adverse health effects of early life exposure to tobacco smoking have been widely reported. In spite of this, the underlying molecular mechanisms of in utero and postnatal exposure to tobacco smoke are only partially understood. Here, we aimed to identify multi-layer molecular signatures associated with exposure to tobacco smoke in these two exposure windows. Methods: We investigated the associations of maternal smoking during pregnancy and childhood secondhand smoke (SHS) exposure with molecular features measured in 1203 European children (mean age 8.1 years) from the Human Early Life Exposome (HELIX) project. Molecular features, covering 4 layers, included blood DNA methylation and gene and miRNA transcription, plasma proteins, and sera and urinary metabolites. Results: Maternal smoking during pregnancy was associated with DNA methylation changes at 18 loci in child blood. DNA methylation at 5 of these loci was related to expression of the nearby genes. However, the expression of these genes themselves was only weakly associated with maternal smoking. Conversely, childhood SHS was not associated with blood DNA methylation or transcription patterns, but with reduced levels of several serum metabolites and with increased plasma PAI1 (plasminogen activator inhibitor-1), a protein that inhibits fibrinolysis. Some of the in utero and childhood smoking-related molecular marks showed dose-response trends, with stronger effects with higher dose or longer duration of the exposure. Conclusion: In this first study covering multi-layer molecular features, pregnancy and childhood exposure to tobacco smoke were associated with distinct molecular phenotypes in children. The persistent and dose-dependent changes in the methylome make CpGs good candidates to develop biomarkers of past exposure. Moreover, compared to methylation, the weak association of maternal smoking in pregnancy with gene expression suggests different reversal rates and a methylation-based memory to past exposures. Finally, certain metabolites and protein markers evidenced potential early biological effects of postnatal SHS, such as fibrinolysis. The study has received funding from the European Community’s Seventh Framework Programme (FP7/2007-206) under grant agreement no. 308333 (the HELIX project); and the H2020-EU.3.1.2. - Preventing Disease Programme under grant agreement no 874583 (the ATHLETE project). BiB received core infrastructure funding from the Wellcome Trust (WT101597MA) and a joint grant from the UK Medical Research Council (MRC) and Economic and Social Science Research Council (ESRC) (MR/N024397/1). INMA data collections were supported by grants from the Instituto de Salud Carlos III, CIBERESP, and the Generalitat de Catalunya-CIRIT. KANC was funded by the grant of the Lithuanian Agency for Science Innovation and Technology (6-04-2014_31V-66). The Norwegian Mother, Father and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research. The RHEA project was financially supported by European projects and the Greek Ministry of Health (Program of Prevention of Obesity and Neurodevelopmental Disorders in Preschool Children, in Heraklion district, Crete, Greece: 2011–2014; “Rhea Plus”: Primary Prevention Program of Environmental Risk Factors for Reproductive Health, and Child Health: 2012–2015). The work was also supported by MICINN (MTM2015-68140-R) and Centro Nacional de Genotipado-CEGEN-PRB2-ISCIII. The CRG/UPF Proteomics Unit is part of the Spanish Infrastructure for Omics Technologies (ICTS OmicsTech), and it is a member of the ProteoRed PRB3 consortium which is supported by grant PT17/0019 of the PE I+D+i 2013-2016 from the Instituto de Salud Carlos III (ISCIII) and ERDF. We acknowledge support from the Spanish Ministry of Science and Innovation through the “Centro de Excelencia Severo Ochoa 2019-2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program.

The enzyme-modified comet assay is widely used for the detection of oxidized DNA lesions. Here we describe for the first time the use of the human alkyladenine DNA glycosylase (hAAG) for the detection of alkylated bases. hAAG was titrated using untreated and methyl methanesulfonate (MMS)-treated TK-6 cells. The hAAG-modified comet assay was compared to the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay, widely used to detect oxidized lesions but that also detects ring-opened purines derived from some alkylated lesions, using cells treated with potassium bromate (oxidizing agent) or MMS. Moreover, neutral and alkaline lysis conditions were used to determine the nature of detected lesions. When alkaline lysis was employed (condition normally used), the level of hAAG-sensitive sites was higher than the Fpg-sensitive sites in MMS-treated cells and hAAG, unlike Fpg, did not detect oxidized bases. After neutral lysis, Fpg did not detect MMS-induced lesions; however, results obtained with hAAG remained unchanged. As expected, Fpg detected oxidized purines and imidazole ring-opened purines, derived from N7-methylguanines under alkaline conditions. It seems that hAAG detected N7-methylguanines, the ring-opened purines derived at high pH, and 3-methlyladenines. Specificity of hAAG towards different DNA lesions was evaluated using a multiplex oligonucleotide-cleavage assay, confirming the ability of hAAG to detect ethenoadenines and hypoxanthine. The hAAG-modified comet assay is a new tool for the detection of alkylated bases.

Attention-deficit/hyperactivity disorder (ADHD) is the most common neuropsychiatric disorder in childhood affecting 5-6% of children, and is a major global health concern, which seems to increase in magnitude. The etiology of ADHD is still poorly understood, however; there are indications of genetic as well as environmental and epigenetic factors contributing to the development of the disorder. The objectives of this study was i) to identify potential ADHD candidate genes; ii) to explore spatial and temporal transcriptional fluctuation of the identified ADHD candidate genes in normal developing human brain tissues, and iii) to identify miRNAs regulating the identified ADHD candidate genes and explore how these miRNAs are expressed in normal developing human brain tissues. From search in literature and publicly available databases, we identified 103 shared potential ADHD candidate genes. These genes were expressed and enriched in several human brain regions and developmental stages. Clustering analysis of these genes based on their expression levels showed a clear difference between fetal stage and the other developmental stages. There was no clear gender or brain region differences between samples. Further, functional analysis of these genes revealed that they participate in a variety of different and widely distributed functional pathways implicated with ADHD. From miRNA-target prediction analysis, we identified twenty miRNAs regulating the identified 103 genes, and the expression pattern of these miRNAs was developmental stage dependent. These miRNAs were enriched in functional pathways and disease ontologies relevant to neurodevelopment. The knowledge of the expression pattern of potential ADHD candidate genes and miRNAs, which regulate these genes across different stages of brain development, is essential for understanding normal brain development and subsequent disease development of the brain. In addition, identification of miRNA-regulated ADHD candidate genes can be used to develop blood-based molecular markers to be investigated in future studies of ADHD patients.

BackgroundMicroRNAs (miRNAs) have been linked to several diseases and to regulation of almost every biological process. This together with their stability while freely circulating in blood suggests that they could serve as minimal-invasive biomarkers for a wide range of diseases. Successful miRNA-based biomarker discovery in plasma is dependent on controlling sources of preanalytical variation, such as cellular contamination and hemolysis, as they can be major causes of altered miRNA expression levels. Analysis of plasma quality is therefore a crucial step for the best output when searching for novel miRNA biomarkers.MethodsPlasma quality was assessed by three different methods in samples from mother-child duos (maternal and cord blood, N = 2x38), with collection and storage methods comparable to large cohort study biobanks. Total RNA was isolated and the expression profiles of 201 miRNAs was obtained by qPCR to identify differentially expressed miRNAs in cord and maternal plasma samples.ResultsAll three methods for quality assurance indicate that the plasma samples used in this study are of high quality with very low levels of contamination, suitable for analysis of circulating miRNAs. We identified 19 significantly differentially expressed miRNAs between cord and maternal plasma samples (paired t-tests, FDR±1.5), and we observed low correlation of miRNA transcript levels between cord and maternal samples throughout our dataset.ConclusionsOur findings suggest that good quality plasma samples suitable for miRNA profiling can be achieved from samples collected and stored by large biobanks. Incorporation of extensive quality control measures, such as those established here, would be beneficial for future projects. The overall low correlation of miRNA expression between cord and maternal samples is an interesting observation, and promising for our future studies on identification of miRNA-based biomarkers in cord blood plasma, considering that these samples were collected at term and some exchange of blood components between cord and maternal blood frequently occur.

Even if the comet assay has been widely used for decades, there is still a need for controlled studies and good mathematical models to assess the variability of the different versions of this assay and in particular to assess potential intra-experimental variability of the high-throughput comet assay. To address this point, we further validate a high-throughput comet assay that uses hydrophilic polyester film (Gelbond®). Experiments were performed using human peripheral blood mononuclear cells (PBMC) either untreated or treated with different concentration of MMS (methyl methanesulfonate). A positive control for the Fpg (Formamidopyrimidine DNA glycosylase)-modified comet assay (Ro 19-8022 with light) was also included. To quantify the sources of variability of the assay, including intradeposit variability, instead of summarizing DNA damage on 50 cells from a deposit by the mean or median of their percentage DNA tail, we analyzed all logit-transformed data with a linear mixed model. The main source of variation in our experimental data is between cells within the same deposit, suggesting genuine variability in the response of the cells rather than variation caused by technical treatment of cell samples. The second source of variation is the inter-experimental variation (day-to-day experiment); the coefficient of this variation for the control was 13.6%. The variation between deposits in the same experiment is negligible. Moreover, there is no systematic bias because of the position of samples on the Gelbond® film nor the position of the films in the electrophoresis tank. This high-throughput comet assay is thus reliable for various applications. Environ. Mol. Mutagen. 59:595-602, 2018. © 2018 Wiley Periodicals, Inc.

The biological effects of gamma radiation may exert damage beyond that of the individual through its deleterious effects on reproductive function. Impaired reproductive performance can result in reduced population size over consecutive generations. In a continued effort to investigate reproductive and heritable effects of ionizing radiation, we recently demonstrated adverse effects and genomic instability in progeny of parents exposed to gamma radiation. In the present study, genotoxicity and effects on the reproduction following subchronic exposure during a gametogenesis cycle to 60Co gamma radiation (27 days, 8.7 and 53 mGy/h, total doses 5.2 and 31 Gy) were investigated in the adult wild-type zebrafish (Danio rerio). A significant reduction in embryo production was observed one month after exposure in the 53 mGy/h exposure group compared to control and 8.7 mGy/h. One year later, embryo production was significantly lower in the 53 mGy/h group compared only to control, with observed sterility, accompanied by a regression of reproductive organs in 100% of the fish 1.5 years after exposure. Histopathological examinations revealed no significant changes in the testis in the 8.7 mGy/h group, while in 62.5% of females exposed to this dose rate the oogenesis was found to be only at the early previtellogenic stage. The DNA damage determined in whole blood, 1.5 years after irradiation, using a high throughput Comet assay, was significantly higher in the exposed groups (1.2 and 3-fold increase in 8.7 and 53 mGy/h females respectively; 3-fold and 2-fold increase in 8.7 and 53 mGy/h males respectively) compared to controls. A significantly higher number of micronuclei (4–5%) was found in erythrocytes of both the 8.7 and 53 mGy/h fish compared to controls. This study shows that gamma radiation at a dose rate of ≥ 8.7 mGy/h during gametogenesis causes adverse reproductive effects and persistent genotoxicity (DNA damage and increased micronuclei) in adult zebrafish. Gamma irradiation during gametogenesis in young adult zebrafish causes persistent genotoxicity and adverse reproductive effects

The alkaline comet assay and a cell-free system were used to characterise DNA lesions induced by treatment with glycidamide (GA), a metabolite of the food contaminant acrylamide. DNA lesions induced by GA were sensitively detected when the formamidopyrimidine-DNA-glycosylase (Fpg) enzyme was included in the comet assay. We used LC-MS to characterise modified bases from GA-treated naked DNA with and without subsequent Fpg treatment. N7-GA-Guanine and N3-GA-Adenine aglycons were detected in the supernatant showing some depurination of adducted bases; treatment of naked DNA with Fpg revealed no further increase in the adduct yield nor occurrence of other adducted nucleobases. We treated human lymphocytes with GA and found large differences in DNA lesion levels detected with Fpg, depending on the duration and the pH of the lysis step. These lysis-dependent variations in GA-induced Fpg sensitive sites paralleled those observed after treatment of cells with methyl methane sulfonate (MMS). On the other hand, oxidative lesions (8-oxoGuanine) induced by a photoactive compound (Ro 12-9786) plus light, and also DNA strand breaks induced by X-rays, were detected largely independently of the lysis conditions. The results suggest that the GA-induced lesions are predominantly N7-GA-dG adducts slowly undergoing imidazole ring opening at pH 10 as in the standard lysis procedure; such structures are substrate for Fpg leading to strand breaks. The data suggest that the characteristic alkaline lysis dependence of some DNA lesions may be used to study specific types of DNA modifications. The comet assay is increasingly used in regulatory testing of chemicals; in this context, lysis-dependent variations represent a novel approach to obtain insight in the molecular nature of a genotoxic insult.

Background: Telomere length is a molecular marker of biological aging. Objective: Here we investigated whether early-life exposure to residential air pollution was associated with leukocyte telomere length (LTL) at 8 y of age. Methods: In a multicenter European birth cohort study, HELIX (Human Early Life Exposome) ([Formula: see text]), we estimated prenatal and 1-y childhood exposure to nitrogen dioxide ([Formula: see text]), particulate matter with aerodynamic diameter [Formula: see text] ([Formula: see text]), and proximity to major roads. Average relative LTL was measured using quantitative real-time polymerase chain reaction (qPCR). Effect estimates of the association between LTL and prenatal, 1-y childhood air pollution, and proximity to major roads were calculated using multiple linear mixed models with a random cohort effect and adjusted for relevant covariates. Results: LTL was inversely associated with prenatal and 1-y childhood [Formula: see text] and [Formula: see text] exposures levels. Each standard deviation (SD) increase in prenatal [Formula: see text] was associated with a [Formula: see text] (95% CI: [Formula: see text], [Formula: see text]) change in LTL. Prenatal [Formula: see text] was nonsignificantly associated with LTL ([Formula: see text] per SD increase; 95% CI: [Formula: see text], 0.6). For each SD increment in 1-y childhood [Formula: see text] and [Formula: see text] exposure, LTL shortened by [Formula: see text] (95% CI: [Formula: see text], [Formula: see text]) and [Formula: see text] (95% CI: [Formula: see text], 0.1), respectively. Each doubling in residential distance to nearest major road during childhood was associated with a 1.6% (95% CI: 0.02, 3.1) lengthening in LTL. Conclusion: Lower exposures to air pollution during pregnancy and childhood were associated with longer telomeres in European children at 8 y of age. These results suggest that reductions in traffic-related air pollution may promote molecular longevity, as exemplified by telomere length, from early life onward. https://doi.org/10.1289/EHP4148. The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) under grant agreement no. 308333 (the HELIX project) and from Instituto de Salud Carlos III (PI15/00118, including The European Regional Development Fund (FEDER)). T.S.N. was funded by the EU Program “Ideas” (ERC-2012-StG 310898). ISGlobal is a member of the Centres de Recerca de Catalunya (CERCA) Programme, Generalitat de Catalunya. We are grateful to all the participating families in the six countries who took part in this study.

The comet assay, also called single cell gel electrophoresis, is a sensitive, rapid and low-cost technique for quantifying and analysing DNA damage and repair at the level of individual cells. The assay itself can be applied on virtually any cell type derived from different organs and tissues of eukaryotic organisms. Although it is mainly used on human cells, the assay has applications also in the evaluation of DNA damage in yeast, plant and animal cells. Therefore, the purpose of this review is to give an extensive overview on the usage of the comet assay in animal models from invertebrates to vertebrates, covering both terrestrial and water biota. The comet assay is used in a variety of invertebrate species since they are regarded as interesting subjects in ecotoxicological research due to their significance in ecosystems. Hence, the first part of the review (Part 1) will discuss the application of the comet assay in invertebrates covering protozoans, platyhelminthes, planarians, cnidarians, molluscs, annelids, arthropods and echinoderms. Besides a large number of animal species, the assay is also performed on a variety of cells, which includes haemolymph, gills, digestive gland, sperm and embryo cells. The mentioned cells have been used for the evaluation of a broad spectrum of genotoxic agents both in vitro and in vivo. Moreover, the use of invertebrate models and their role from an ecotoxicological point of view will also be discussed as well as the comparison of the use of the comet assay in invertebrate and human models. Since the comet assay is still developing, its increasing potential in assessing DNA damage in animal models is crucial especially in the field of ecotoxicology and biomonitoring at the level of different species, not only humans. This work was supported by the European Cooperation in Science and Technology (CA COST Action CA15132 – The comet assay as a human biomonitoring tool (hCOMET)). info:eu-repo/semantics/publishedVersion

Characterization of the "exposome", the set of all environmental factors that one is exposed to from conception onwards, has been advocated to better understand the role of environmental factors on chronic diseases. Here, we aimed to describe the early-life exposome. Specifically, we focused on the correlations between multiple environmental exposures, their patterns and their variability across European regions and across time (pregnancy and childhood periods). We relied on the Human Early-Life Exposome (HELIX) project, in which 87 environmental exposures during pregnancy and 122 during the childhood period (grouped in 19 exposure groups) were assessed in 1301 pregnant mothers and their children at 6-11 years in 6 European birth cohorts. Some correlations between exposures in the same exposure group reached high values above 0.8. The median correlation within exposure groups was >0.3 for many exposure groups, reaching 0.69 for water disinfection by products in pregnancy and 0.67 for the meteorological group in childhood. Median correlations between different exposure groups rarely reached 0.3. Some correlations were driven by cohort-level associations (e.g. air pollution and chemicals). Ten principal components explained 45% and 39% of the total variance in the pregnancy and childhood exposome, respectively, while 65 and 90 components were required to explain 95% of the exposome variability. Correlations between maternal (pregnancy) and childhood exposures were high (>0.6) for most exposures modeled at the residential address (e.g. air pollution), but were much lower and even close to zero for some chemical exposures. In conclusion, the early life exposome was high dimensional, meaning that it cannot easily be measured by or reduced to fewer components. Correlations between exposures from different exposure groups were much lower than within exposure groups, which have important implications for co-exposure confounding in multiple exposure studies. Also, we observed the early life exposome to be variable over time and to vary by cohort, so measurements at one time point or one place will not capture its complexities. This work was supported by the European Commission Seventh Framework Programme (FP7/2007–2013) [grant number: 308333–the HELIX project]. INMA data collections were supported by grants from the Instituto de Salud Carlos III, CIBERESP, and the Generalitat de Catalunya-CIRIT. KANC was funded by the grant of the Lithuanian Agency for Science Innovation and Technology (6-04-2014_31V-66). The Norwegian Mother and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research, NIH/NIEHS (contract no N01-ES-75558), NIH/NINDS (grant no.1 UO1 NS 047537-01 and grant no.2 UO1 NS 047537-06A1). The Rhea project was financially supported by European projects (EU FP6-2003-Food-3-NewGeneris, EU FP6. STREP Hiwate, EU FP7 ENV.2007.1.2.2.2. Project No 211250 Escape, EU FP7-2008-ENV-1.2.1.4 Envirogenomarkers, EU FP7-HEALTH-2009-single stage CHICOS, EU FP7 ENV.2008.1.2.1.6. Proposal No 226285 ENRIECO, EUFP7-HEALTH-2012 Proposal No 308333 HELIX, FP7 European Union project, No. 264357 MeDALL), and the Greek Ministry of Health (Program of Prevention of obesity and neurodevelopmental disorders in preschool children, in Heraklion district, Crete, Greece: 2011–2014; “Rhea Plus”: Primary Prevention Program of Environmental Risk Factors for Reproductive Health, and Child Health: 2012–15).

Background: Prenatal exposure to air pollution has been associated with childhood respiratory disease and other adverse outcomes. Epigenetics is a suggested link between exposures and health outcomes. Objectives: We aimed to investigate associations between prenatal exposure to particulate matter (PM) with diameter

Background: Harmonized data describing simultaneous exposure to a large number of environmental contaminants in-utero and during childhood is currently very limited. Objectives: To characterize concentrations of a large number of environmental contaminants in pregnant women from Europe and their children, based on chemical analysis of biological samples from mother-child pairs. Methods: We relied on the Early-Life Exposome project, HELIX, a collaborative project across six established population-based birth cohort studies in Europe. In 1301 subjects, biomarkers of exposure to 45 contaminants (i.e. organochlorine compounds, polybrominated diphenyl ethers, per- and polyfluoroalkyl substances, toxic and essential elements, phthalate metabolites, environmental phenols, organophosphate pesticide metabolites and cotinine) were measured in biological samples from children (6-12 years) and their mothers during pregnancy, using highly sensitive biomonitoring methods. Results: Most of the exposure biomarkers had high detection frequencies in mothers (35 out of 45 biomarkers with >90% detected) and children (33 out of 45 biomarkers with >90% detected). Concentrations were significantly different between cohorts for all compounds, and were generally higher in maternal compared to children samples. For most of the persistent compounds the correlations between maternal and child concentrations were moderate to high (Spearman Rho > 0.35), while for most non-persistent compounds correlations were considerably lower (Spearman Rho < 0.15). For mercury, PFOS and PFOA a considerable proportion of the samples of both mothers and their children exceeded the HBM I value established by The Human Biomonitoring Commission of the German Federal Environment Agency. Discussion: Although not based on a representative sample, our study suggests that children across Europe are exposed to a wide range of environmental contaminants in fetal life and childhood including many with potential adverse effects. For values exceeding the HBM I value identification of specific sources of exposure and reducing exposure in an adequate way is recommended. Considerable variability in this "chemical exposome" was seen between cohorts, showing that place of residence is a strong determinant of one's personal exposome. This extensive dataset comprising >100,000 concentrations of environmental contaminants in mother-child pairs forms a unique possibility for conducting epidemiological studies using an exposome approach. The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007–2013) under grant agreement no 308333 – the HELIX project. Dr. Regina Grazuleviciene received the grant of the Lithuanian Agency for Science Innovation and Technology (No. 31V-77). Dr. Maribel Casas received funding from Instituto de Salud Carlos III (Ministry of Economy and Competitiveness) (MS16/00128). Rosie McEachan and John Wright were supported by the NIHR CLAHRC Yorkshire and Humber (IS-CLA-0113-10020). www.clahrc-yh.nihr.ac.uk. The INMA (Environment and Childhood) Sabadell cohort and biomarker measurements were funded by grants from Instituto de Salud Carlos III (Red INMA G03/176; CB06/02/0041; PI041436; PI081151 incl. FEDER funds; PI12/01890 incl. FEDER funds; CP13/00054 incl. FEDER funds), CIBERESP, Generalitat de Catalunya-CIRIT 1999SGR 00241, Generalitat de Catalunya-AGAUR (2009 SGR 501, 2014 SGR 822), Fundació La marató de TV3 (090430), Spanish Ministry of Economy and Competitiveness (SAF2012-32991 incl. FEDER funds). The Norwegian Mother and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research, NIH/NINDS (grant no.1 UO1 NS 047537-01 and grant no.2 UO1 NS 047537-06A1). The REAH cohort was financially supported by European projects (EU FP6-2003-Food-3-NewGeneris, EU FP6. STREP Hiwate, EU FP7 ENV.2007.1.2.2.2. Project No 211250 Escape, EU FP7-2008-ENV-1.2.1.4 Envirogenomarkers, EU FP7-HEALTH-2009- single stage CHICOS, EU FP7 ENV.2008.1.2.1.6. Proposal No 226285 ENRIECO, EU- FP7- HEALTH-2012 Proposal No 308333 HELIX), the Greek Ministry of Health (Program of Prevention of obesity and neurodevelopmental disorders in preschool children, in Heraklion district, Crete, Greece: 2011–2014; “Rhea Plus”: Primary Prevention Program of Environmental Risk Factors for Reproductive Health, and Child Health: 2012–15).

Telomere length and mitochondrial DNA content are considered biomarkers of cellular aging, oxidative stress, and inflammation, but there is almost no information on their association with tobacco smoke exposure in fetal and early life. The aim of this study was to assess whether prenatal and childhood tobacco exposure were associated with leukocyte telomere length (LTL) and mitochondrial DNA (mtDNA) content in children. As part of a multi-centre European birth cohort study HELIX (Human Early-Life Exposome) (n = 1396) we assessed maternal smoking status during pregnancy through questionnaires, and through urinary cotinine levels that were then used to classify women as not exposed to smoking (50 mu g/L). When the children were around 8 years of age (range: 5.4-12.0 years), childhood SHS tobacco smoke exposure was assessed through an extensive questionnaire and through measurements of urinary cotinine (3.03 mu g/L detected). Leukocyte mtDNA content and LTL were measured in the children at 8 years employing real time polymerase chain reaction (qPCR). Effect estimates were calculated using multivariate linear regression models for prenatal and childhood exposures adjusted for potential confounders. Maternal cotinine levels indicative of SHS exposure during pregnancy were associated with a decrease of 3.90% in LTL in children (95% CI: -6.68, -0.91), compared with nonsmoking, whereas the association for maternal cotinine levels indicative of active smoking did not reach statistical significance (-3.24%; 95% CI: -6.59, 0.21). Childhood SHS tobacco exposure was not associated with LTL in children. Global SHS exposure during childhood was associated with an increase of 3.51% (95% CI: 0.78, 6.27) in mtDNA content. Our findings suggest that tobacco smoke exposure during pregnancy, even at SHS levels, may accelerate telomere shortening in children and thus induce biological aging from an early age. (C) 2019 Elsevier B.V. All rights reserved.

The toxicity of some per- and polyfluoroalkyl substances (PFASs), such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) has been studied thoroughly, showing that systemic PFASs targets the lungs. However, regulators lack data to assess the impact of other PFASs on the lungs and alternative methods to test substances for lung toxicity are needed. We combined two in vitro models to assess toxicity to the respiratory system; i) a lung surfactant (LS) function assay to assess the acute inhalation toxicity potential, and ii) a cell model with human bronchial epithelial cells to study pro-inflammatory potential and modulation of inflammatory responses. We tested salts of four PFASs: perfluorobutane sulfonate (PFBS), perfluorohexane sulfonate (PFHxS), PFOS, and PFOA as well as the fluorotelomer 8:2 FTOH. The results show that PFHxS, PFOA and PFOS can inhibit LS function. High PFOS concentrations induced a pro-inflammatory response, measured as increased IL-1α/β release. Moderate concentrations of PFOS suppressed release of the chemokines CXCL8 and CXCL10, whereas both PFOS and PFOA stimulated the release of the pro-inflammatory cytokine IL-1β in immune stimulated human bronchial epithelial cells. These findings support the concern that some PFASs may increase the risk of acute lung toxicity and of airway infections.

There is increase concerns over child behavioral problems due to changing environments. Children are typically exposed to a long list of industrial chemicals that are neurotoxic but also to benefic...

Background: Several environmental contaminants have been identified as possibly influencing birth weight, mainly from single exposure studies. The Exposome paradigm offers perspectives to avoid sel...

Occupational exposure to diesel exhaust may cause lung cancer in humans. Mechanisms include DNA-damage and inflammatory responses. Here, the potential of NIST SRM2975 diesel exhaust particles (DEP) to transform human bronchial epithelial cells (HBEC3) in vitro was investigated. Long-term exposure of HBEC3 to DEP led to increased colony growth in soft agar. Several DEP-transformed cell lines were established and based on the expression of epithelial-to-mesenchymal-transition (EMT) marker genes, one of them (T2-HBEC3) was further characterized. T2-HBEC3 showed a mesenchymal/fibroblast-like morphology, reduced expression of CDH1, and induction of CDH2 and VIM. T2-HBEC3 had reduced migration potential compared with HBEC3 and little invasion capacity. Gene expression profiling showed baseline differences between HBEC3 and T2-HBEC3 linked to lung carcinogenesis. Next, to assess differences in sensitivity to DEP between parental HBEC3 and T2-HBEC3, gene expression profiling was carried out after DEP short-term exposure. Results revealed changes in genes involved in metabolism of xenobiotics and lipids, as well as inflammation. HBEC3 displayed a higher steady state of IL1B gene expression and release of IL-1β compared with T2-HBEC3. HBEC3 and T2-HBEC3 showed similar susceptibility towards DEP-induced genotoxic effects. Liquid-chromatography-tandem-mass-spectrometry was used to measure secretion of eicosanoids. Generally, major prostaglandin species were released in higher concentrations from T2-HBEC3 than from HBEC3 and several analytes were altered after DEP-exposure. In conclusion, long-term exposure to DEP-transformed human bronchial epithelial cells in vitro. Differences between HBEC3 and T2-HBEC3 regarding baseline levels and DEP-induced changes of particularly CYP1A1, IL-1β, PGE2, and PGF2α may have implications for acute inflammation and carcinogenesis.

Among aquatic organisms, invertebrate species such as the freshwater crustacean Daphnia magna are believed to be sensitive to gamma radiation, although information on responses at the individual, biochemical and molecular level is scarce. Following gamma radiation exposure, biological effects are attributed to the formation of free radicals, formation of reactive oxygen species (ROS) and subsequently oxidative damage to lipids, proteins and DNA in exposed organisms. Thus, in the present study, effects and modes of action (MoA) have been investigated in D. magna exposed to gamma radiation (dose rates: 0.41, 1.1, 4.3, 10.7, 42.9 and 106 mGy/h) after short-term exposure (24 and 48 h). Several individual, cellular and molecular endpoints were addressed, such as ROS formation, lipid peroxidation, DNA damage and global transcriptional changes. The results showed that oxidative stress is one of the main toxic effects in gamma radiation exposed D. magna, mediated by the dose-dependent increase in ROS formation and consequently oxidative damage to lipids and DNA over time. Global transcriptional analysis verified oxidative stress as one of the main MoA of gamma radiation at high dose rates, and identified a number of additional MoAs that may be of toxicological relevance. The present study confirmed that acute exposure to gamma radiation caused a range of cellular and molecular effects in D. magna exposed to intermediate dose rates, and highlights the need for assessing effects at longer and more environmentally relevant exposure durations in future studies.

Background: Several environmental contaminants were reported to possibly influence fetal growth, generally from single exposure family studies, which are prone to publication bias. The exposome paradigm offers perspectives to avoid selective reporting of findings and to control for confounding by co-exposures. Objectives: We aimed to assess the association of the pregnancy chemical and external exposomes with fetal growth. Methods: Within HELIX project, 131 prenatal exposures were assessed using biomarkers and environmental models in 1,287 mother-child pairs from six European cohorts. We investigated their associations with fetal growth using a deletion-substitution-addition (DSA) algorithm considering all exposures simultaneously, and an exposome-wide association study (ExWAS) considering each exposure independently. We corrected for exposure misclassification, and tested for exposure-exposure and sex-exposure interactions. Findings: Lead blood level was the only exposure associated with fetal growth in the DSA model (97g weight decrease for each doubling in lead concentration). No exposure passed the multiple testing-corrected significance threshold of ExWAS; without multiple testing correction, this model was in favor of a negative association of fine particulate matter concentration with birth weight; and of a positive sex-specific association of parabens with birth weight in boys only. No two-way interaction between exposure variables was identified. Interpretation: This first large-scale exposome study of fetal growth simultaneously considered over 100 environmental exposures. Compared to single exposure studies, our approach allowed making all tests (usually reported in successive publications) explicit. Lead exposure is still a health concern in Europe and parabens health effects warrant further investigation. Funding Statement: FP7/2007-2013, HELIX project (no 308333). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The study was approved by the relevant ethical committees from each country and an informed consent form was assigned from all participants, or the parents of the children.

Background: Bulky DNA adducts reflect genotoxic exposures, have been associated with lower birth weight, and may predict cancer risk. Objective: We selected factors known or hypothesized to affect in utero adduct formation and repair and examined their associations with adduct levels in neonates. Methods: Pregnant women from Greece, Spain, England, Denmark, and Norway were recruited in 2006–2010. Cord blood bulky DNA adduct levels were measured by the 32P-postlabeling technique (n = 511). Diet and maternal characteristics were assessed via questionnaires. Modeled exposures to air pollutants and drinking-water disinfection by-products, mainly trihalomethanes (THMs), were available for a large proportion of the study population. Results: Greek and Spanish neonates had higher adduct levels than the northern European neonates [median, 12.1 (n = 179) vs. 6.8 (n = 332) adducts per 108 nucleotides, p < 0.001]. Residence in southern European countries, higher maternal body mass index, delivery by cesarean section, male infant sex, low maternal intake of fruits rich in vitamin C, high intake of dairy products, and low adherence to healthy diet score were statistically significantly associated with higher adduct levels in adjusted models. Exposure to fine particulate matter and nitrogen dioxide was associated with significantly higher adducts in the Danish subsample only. Overall, the pooled results for THMs in water show no evidence of association with adduct levels; however, there are country-specific differences in results with a suggestion of an association in England. Conclusion: These findings suggest that a combination of factors, including unknown country-specific factors, influence the bulky DNA adduct levels in neonates. Citation: Pedersen M, Mendez MA, Schoket B, Godschalk RW, Espinosa A, Landström A, Villanueva CM, Merlo DF, Fthenou E, Gracia-Lavedan E, van Schooten FJ, Hoek G, Brunborg G, Meltzer HM, Alexander J, Nielsen JK, Sunyer J, Wright J, Kovács K, de Hoogh K, Gutzkow KB, Hardie LJ, Chatzi L, Knudsen LE, Anna L, Ketzel M, Haugen M, Botsivali M, Nieuwenhuijsen MJ, Cirach M, Toledano MB, Smith RB, Fleming S, Agramunt S, Kyrtopoulos SA, Lukács V, Kleinjans JC, Segerbäck D, Kogevinas M. 2015. Environmental, dietary, maternal, and fetal predictors of bulky DNA adducts in cord blood: a European mother–child study (NewGeneris). Environ Health Perspect 123:374–380; http://dx.doi.org/10.1289/ehp.1408613

The alkaline comet assay, in vivo and in vitro, is currently used in several areas of research and in regulatory genotoxicity testing. Several efforts have been made in order to decrease the inter-experimental and inter-laboratory variability and increase the reliability of the assay. In this regard, lysis conditions are considered as one of the critical variables and need to be further studied. Here, we tested different times of lysis (from no lysis to 1 week) and two different lysis solutions in human lymphoblast (TK6) cells unexposed or exposed to X-rays. Similar % tail DNA values were obtained independently of the time of lysis employed for every X-ray dose tested and both lysis solutions. These results, taken together with our previous ones with methyl methanesulfonate and H2O2, which showed clear lysis-time dependence, support that the influence of the lysis time in the comet assay results depends on the type of lesion being detected; some DNA lesions may spontaneously give rise to apurinic or apyrimidinic (AP) sites during the lysis period, which can be converted into strand breaks detectable with the comet assay. Testing different times of lysis would be useful to increase the sensitivity of the comet assay and to ensure the detection of DNA lesions of an unknown compound, thereby providing some insight into the chemical nature of the lesions induced. However, the same lysis conditions (i.e. lysis time and lysis solution) should be used when comparing results between different experiments or laboratories.

Maternal diet can result in exposure to environmental contaminants including dioxins which may influence foetal growth. We investigated the association between maternal diet and birth outcomes by defining a dioxin-rich diet. We used validated food frequency questionnaires to assess the diet of pregnant women from Greece, Spain, United Kingdom, Denmark and Norway and estimated plasma dioxin-like activity by the Dioxin-Responsive Chemically Activated LUciferase eXpression (DR-CALUX?) bioassay in 604 maternal blood samples collected at delivery. We applied reduced rank regression to identify a dioxin-rich dietary pattern based on dioxin-like activity (DR-CALUX?) levels in maternal plasma, and calculated a dioxin-diet score as an estimate of adherence to this dietary pattern. In the five country population, dioxin-diet score was characterised by high consumption of red and white meat, lean and fatty fish, low-fat dairy and low consumption of salty snacks and high-fat cheese, during pregnancy. The upper tertile of the dioxin-diet score was associated with a change in birth weight of -121g (95% confidence intervals: -232, -10g) compared to the lower tertile after adjustment for confounders. A small non-significant reduction in gestational age was also observed (-1.4days, 95% CI: -3.8, 1.0days). Our results suggest that maternal diet might contribute to the exposure of the foetus to dioxins and dioxin-like compounds and may be related to reduced birth weight. More studies are needed to develop updated dietary guidelines for women of reproductive age, aiming to the reduction of dietary exposure to persistent organic pollutants as dioxins and dioxin-like compounds.