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Background: Residential green space may improve human health, for example by promoting physical activity and by reducing stress. Conversely, residential green space may increase stress by emitting aeroallergens and exacerbating allergic disease. Here we examine impacts of exposure to residential green space on distress in the susceptible subpopulation of adults sensitized to tree pollen allergens. Methods: In a panel study of 88 tree pollen allergy patients we analyzed self-reported mental health (GHQ-12), perceived presence of allergenic trees (hazel, alder, birch) near the residence and residential green space area within 1 km distance [high (>= 3 m) and low (< 3 m) green]. Results were adjusted for patients' background data (gender, age, BMI, smoking status, physical activity, commuting distance, education level, allergy medication use and chronic respiratory problems) and compared with distress in the general population (N = 2467). Results: Short-term distress [mean GHQ-12 score 2.1 (95% confidence interval 1.5-2.7)] was higher in the study population than in the general population [1.5 (1.4-1.7)]. Residential green space had protective effects against short-term distress [high green, per combined surface area of 10 ha: adjusted odds ratio OR = 0.94 (95% confidence interval 0.90-0.99); low green, per 10 ha: OR = 0.85 (0.78-0.93)]. However, distress was higher in patients who reported perceived presence of allergenic trees near their residence [present vs. absent: OR = 2.04 (1.36-3.07)]. Conclusions: Perceived presence of allergenic tree species in the neighbourhood of the residence of tree pollen allergy patients modulates the protective effect of residential green space against distress during the airborne tree pollen season. The RespirIT study has been supported by a project grant from the Belgian Science Policy Office BELSPO (grant nr. BR/154/A1/RespirIT). The GHQ-12 questionnaire was used under license from GL Assessment Ltd. Aerts, R (reprint author), Sciensano, Juliette Wytsmanstr 14, BE-1050 Brussels, Belgium. raf.aerts@sciensano.be

Particle transfer across the placenta has been suggested but to date, no direct evidence in real-life, human context exists. Here we report the presence of black carbon (BC) particles as part of combustion-derived particulate matter in human placentae using white-light generation under femtosecond pulsed illumination. BC is identified in all screened placentae, with an average (SD) particle count of 0.95 × 104 (0.66 × 104) and 2.09 × 104 (0.9 × 104) particles per mm3 for low and high exposed mothers, respectively. Furthermore, the placental BC load is positively associated with mothers’ residential BC exposure during pregnancy (0.63–2.42 µg per m3). Our finding that BC particles accumulate on the fetal side of the placenta suggests that ambient particulates could be transported towards the fetus and represents a potential mechanism explaining the detrimental health effects of pollution from early life onwards., Exposure to air pollution during pregnancy has been associated with impaired birth outcomes. Here, Bové et al. report evidence of black carbon particle deposition on the fetal side of human placentae, including at early stages of pregnancy, suggesting air pollution could affect birth outcome through direct effects on the fetus.

Background Studies investigating short-term exposure to ambient air pollution and heart rate variability (HRV) suggest that particulate matter (PM) exposure is associated with reductions in measures of HRV. Mitochondria are sensitive to PM exposure and may represent a biologically relevant underlying mechanism. However, evidence in children is lacking. Objectives Here we examine whether PM has an influence on children's HRV and evaluate whether mitochondrial DNA content (mtDNAc) reflects individual susceptibility. Methods Within a panel study in primary school children (aged 9–12 years), we measured HRV in a subset of 60 children on three different days during school-time using four indicators: normal-to-normal intervals (SDNN), square root of mean squared difference of normal-to-normal intervals (rMSSD), high frequency (HF), and low frequency (LF). This resulted in a total number of 150 visits (median number of visits per child: 2.5/child). MtDNAc was measured using qPCR in buccal cells. We measured recent PM exposure at the school. Residential 24-hour mean exposure to PM was modelled with a high resolution spatial temporal model. Mixed-effects models were used to estimate the association between HRV and recent PM exposure and potential effect-modification by mtDNAc. Results Children were on average [SD] 9.9 [1.2] years and comprised 39 girls. Median [25th–75th] recent outdoor PM2.5 and PM10 exposure at school was 6.20 [2.8–12.8] μg/m3 and 29.3 [24.7–42.0] μg/m3, respectively. In children with low mtDNAc (25th percentile), we observed for each 10 μg/m3 increment in recent PM2.5 exposure a lowering in the LF parameter with 9.76% (95% CI: −16.9 to −1.99%, p = 0.02; pint = 0.007). Children with high mtDNAc did not show this association. For PM10 exposure, we observed an inverse association with three HRV indicators in children with low mtDNAc: −2.24% (95% CI: −4.27 to −0.16%; p = 0.04; pint = 0.02) for SDNN, −5.67% (95% CI: −10.5 to −0.59%; p = 0.03; pint = 0.04) for HF and −6.64% (95% CI: −10.7 to −2.38%; p = 0.003; pint = 0.005) for LF. Conclusions HRV is inversely associated with recent PM air pollution, especially in children with low mtDNAc. Our data revealed that mtDNAc determines susceptibility to adverse autonomic effects of recent PM exposure in children., Highlights • Particulate matter (PM) exposure is associated with lower heart rate variability (HRV). • We found an inverse HRV-PM association in children with low mtDNA content. • The association was not observed in children with high mtDNA content. • Mitochondrial DNA determines susceptibility to adverse autonomic effects of PM.

Background Mental health disorders appear as a growing problem in urban areas. While common mental health disorders are generally linked to demographic and socioeconomic factors, little is known about the interaction with the urban environment. With growing urbanization, more and more people are exposed to environmental stressors potentially contributing to increased stress and impairing mental health. It is therefore important to identify features of the urban environment that affect the mental health of city dwellers. The aim of this study was to define associations of combined long-term exposure to air pollution, noise, surrounding green at different scales, and building morphology with several dimensions of mental health in Brussels. Methods Research focuses on the inhabitants of the Brussels Capital Region older than 15 years. The epidemiological study was carried out based on the linkage of data from the national health interview surveys (2008 and 2013) and specifically developed indicators describing each participant’s surroundings in terms of air quality, noise, surrounding green, and building morphology. These data are based on the geographical coordinates of the participant’s residence and processed using Geographical Information Systems (GIS). Mental health status was approached through several validated indicators: the Symptom Checklist-90-R subscales for depressive, anxiety and sleeping disorders and the 12-Item General Health Questionnaire for general well-being. For each mental health outcome, single and multi-exposure models were performed through multivariate logistic regressions. Results Our results suggest that traffic-related air pollution (black carbon, NO2, PM10) exposure was positively associated with higher odds of depressive disorders. No association between green surrounding, noise, building morphology and mental health could be demonstrated. Conclusions These findings have important implications because most of the Brussel’s population resides in areas where particulate matters concentrations are above the World Health Organization guidelines. This suggests that policies aiming to reduce traffic related-air pollution could also reduce the burden of depressive disorders in Brussels.

Background: Pro-inflammatory conditions such as air pollution might induce biological ageing. However, the available evidence on such an impact in children is still very scarce. We studied in primary schoolchildren the association of ambient residential air pollution exposure with telomere length (TL) and mitochondrial DNA content (mtDNAc), two important targets of the core axis of ageing. Methods: Between 2012 and 2014, buccal TL and mtDNAc were repeatedly assessed using qPCR in 197 Belgian primary schoolchildren (mean age 10.3 years) as part of the COGNAC study. At the child's residence, recent (week), sub-chronic (month) and chronic (year) exposure to nitrogen dioxide (NO2), particulate matter

Introduction: Four epidemiological studies have shown a negative association between prenatal acrylamide exposure and birth size. In order to shed light on the possible underlying mechanism(s), we analysed associations between acrylamide biomarkers and biomarkers related to fetal growth. Methods: In newborns of the ENVIRONAGE birth cohort (n ranges from 215 to 434), we investigated the association between prenatal acrylamide exposure (acrylamide and glycidamide hemoglobin adduct levels in cord blood) and thyroid hormones (TSH, T3, T4 and the ratio of T4 to T3 in cord plasma), insulin-related factors (cord plasma insulin and IGF1, and placental IGF2), neurotrophins (cord plasma BDNF, and placental NGF, NT3 and NT4), and cord plasma homocysteine and progesterone, using multiple linear regression analysis. In addition, we investigated whether the biomarkers mediated the associations between prenatal acrylamide exposure and birth outcomes. Results: We observed lower cord plasma TSH (-10.2% [95% CI: -15.0, -4.3]) and higher placental NGF levels (10.0% [95% CI 3.7, 17.4]) for a twofold increase of acrylamide adducts, a decrease in the ratio of cord plasma free T4 and free T3 with higher acrylamide and glycidamide adducts of -2.9% (95% CI: -5.7, -0.1) and -3.9% (95% CI: -6.2, -1.6) for a twofold increase in acrylamide and glycidamide adduct levels, respectively, and higher cord plasma free T3 with increases in both acrylamide and glycidamide adducts of 2.8% (95% CI: 0.2, 5.6) and 3.6% (95% CI: 0.8, 6.6) for a twofold increase in acrylamide and glycidamide adduct levels, respectively. Additionally, a twofold increase in glycidamide adducts was associated with lower cord plasma insulin levels, particularly among newborns of non-smoking mothers (-11.2% [95% CI: -19.5, -0.1]). Cord plasma insulin seemed to mediate the association between glycidamide adducts and birth weight. Conclusions: A decrease in cord plasma insulin levels may be (a marker of) a mechanism by which gestational acrylamide exposure is associated with decreased fetal growth. The possible health consequences of the associations between gestational acrylamide exposure and thyroid hormones and neurotrophins warrant future study. European Research CouncilEuropean Research Council (ERC)European Commission [ERC-2012-StG310898]; Research Foundation - Flanders (FWO)FWO [1516112N/G.0873.11.N.10, 12J9516N, 1524517N]

Rationale: Ambient air pollution, including black carbon, entails a serious public health risk because of its carcinogenic potential and as climate pollutant. To date, an internal exposure marker for black carbon particles that have cleared from the systemic circulation into the urine does not exist. Objectives: To develop and validate a novel method to measure black carbon particles in a label-free way in urine. Methods: We detected urinary carbon load in 289 children (aged 9–12 yr) using white-light generation under femtosecond pulsed laser illumination. Children’s residential black carbon concentrations were estimated based on a high-resolution spatial temporal interpolation method. Measurements and Main Results: We were able to detect urinary black carbon in all children, with an overall average (SD) of 98.2 × 105 (29.8 × 105) particles/ml. The urinary black carbon load was positively associated with medium-term to chronic (1 mo or more) residential black carbon exposure: +5.33 × 105 particles/ml higher carbon load (95% confidence interval, 1.56 × 105 to 9.10 × 105 particles/ml) for an interquartile range increment in annual residential black carbon exposure. Consistently, children who lived closer to a major road (≤160 m) had higher urinary black carbon load (6.93 × 105 particles/ml; 95% confidence interval, 0.77 × 105 to 13.1 × 105). Conclusions: Urinary black carbon mirrors the accumulation of medium-term to chronic exposure to combustion-related air pollution. This specific biomarker reflects internal systemic black carbon particles cleared from the circulation into the urine, allowing investigators to unravel the complexity of particulate-related health effects. Funded by the European Research Council (ERC-2012-StG 310898), the Interuniversity Attraction Poles Program (P7/05) initiated by the Belgian Science Policy Office, and the Flemish Scientific Research Foundation (G082113/11ZB115N/12R6315N/G073315N).

Introduction: Cardio-metabolic risk factors including insulin levels are at young age barely perceived as harmful, but over time these risk factors may track and lead to higher risk of metabolic syndrome. Studies showed that exposure to air pollution is associated with an increased risk of insulin resistance in childhood. We determined whether the origin of type 2 diabetes can be found in the early childhood by examining the levels of insulin in the neonatal cord blood and whether this can be considered as a disease marker for later life. Methods: In the ENVIRONAGE (ENVIRonmental influence ON early AGEing) birth cohort, we recruited 620 mother-infant pairs between February 2nd 2010 until August 12th 2014 at the East-Limburg Hospital in Genk, Belgium. We investigated in 590 newborns the association between cord plasma insulin levels and exposure to particulate matter (PM2.5 and PM10) and nitrogen dioxide (NO2) in various exposure windows during pregnancy. Trimester-specific air pollutant exposure levels were estimated for each mother's home address using a spatiotemporal model. Results: Cord plasma insulin levels averaged 33.1pmol/L (25-75th percentile: 20.1-53.5), while PM2.5 exposure during pregnancy averaged (SD) 13.7μg/m3 (2.4). Independent of maternal age, newborn's sex, birth weight, gestational age, parity, early-pregnancy BMI, ethnicity, smoking status, time of the day, maternal education, time of delivery, and season of delivery, cord plasma insulin levels increased with 15.8% (95% CI 7.8 to 24.4, p

Background: Thyroid hormones are critical for fetal development and growth. Whether prenatal exposure to fine particle air pollution (PM2.5) affects fetal thyroid function and what the impact is on birth weight in normal healthy pregnancies is not studied yet. Objectives: To study the impact of third trimester PM2.5 exposure on fetal and maternal thyroid hormones and their mediating role on birth weight. Methods: We measured the levels of free thyroid hormones (FT3, FT4) and thyroid stimulating hormone (TSH) in cord blood (n = 499) and maternal blood (n = 431) collected after delivery from mother-child pairs enrolled between February 2010 and June 2014 in the ENVIRONAGE birth cohort with catchment area in the province of Limburg, Belgium. Results: An interquartile range (IQR) increment (+8.2 µg/m3 ) in third trimester PM2.5 exposure was inversely associated with cord blood TSH levels (-11.6%; 95% CI: -21.8, -0.1) and the FT4/FT3 ratio (-62.7%; -91.6, -33.8). A 10th-90th percentile decrease in cord blood FT4 levels was associated with a 56 g decrease in mean birth weight (95% CI: -90, -23). Assuming causality, we estimated that cord blood FT4 mediated 21% (-19 g; 95%CI: -37, -1) of the estimated effect of an IQR increment in third trimester PM2.5 exposure on birth weight. Third trimester PM2.5 exposure was inversely but not significantly associated with maternal blood FT4 levels collected one day after delivery (-4.0%, 95% CI: -8.0, 0.2 for an IQR increment in third trimester PM2.5). Conclusions: In our study population of normal healthy pregnancies, third trimester exposure to PM2.5 air pollution was associated with differences in fetal thyroid hormone levels that may contribute to reduced birth weight. Additional research is needed to confirm our findings in other populations and to evaluate potential consequences later in life. The ENVIRONAGE birth cohort is supported by the European Research Council (ERC-2012-StG.310898), by the Flemish Scientific Fund (FWO, N1516112 / G.0.873.11.N.10) and the Bijzonder Onderzoeksfonds (BOF) of Hasselt University. Karen Vrijens is a postdoctoral fellow of the FWO.

According to the “Developmental Origins of Health and Disease” (DOHaD) concept, the early-life environment is a critical period for fetal programming. Given the epidemiological evidence that air pollution exposure during pregnancy adversely affects newborn outcomes such as birth weight and preterm birth, there is a need to pay attention to underlying modes of action to better understand not only these air pollution-induced early health effects but also its later-life consequences. In this review, we give an overview of air pollution-induced placental molecular alterations observed in the ENVIRONAGE birth cohort and evaluate the existing evidence. In general, we showed that prenatal exposure to air pollution is associated with nitrosative stress and epigenetic alterations in the placenta. Adversely affected CpG targets were involved in cellular processes including DNA repair, circadian rhythm, and energy metabolism. For miRNA expression, specific air pollution exposure windows were associated with altered miR-20a, miR-21, miR-146a, and miR-222 expression. Early-life aging markers including telomere length and mitochondrial DNA content are associated with air pollution exposure during pregnancy. Previously, we proposed the air pollution-induced telomere-mitochondrial aging hypothesis with a direct link between telomeres and mitochondria. Here, we extend this view with a potential co-interaction of different biological mechanisms on the level of placental oxidative stress, epigenetics, aging, and energy metabolism. Investigating the placenta is an opportunity for future research as it may help to understand the fundamental biology underpinning the DOHaD concept through the interactions between the underlying modes of action, prenatal environment, and disease risk in later life. To prevent lasting consequences from early-life exposures of air pollution, policy makers should get a basic understanding of biomolecular consequences and transgenerational risks. This review is supported by grants from the European Research Council (ERC-2012-StG 310898/POC INCALO) and FWO (G082317 N).

The placenta plays a crucial role in fetal growth and development through adaptive responses to perturbations of the maternal environment. We investigated the association between placental 3-nitrotyrosine (3-NTp), a biomarker of oxidative stress, and exposure to air pollutants during various time windows of pregnancy. We measured placental 3-NTp of 330 mother-newborn pairs, enrolled in the ENVIRONAGE birth cohort (2010 to 2013). Daily particulate matter ≤ 2.5 µm (PM2.5), black carbon (BC), and nitrogen dioxide concentrations were interpolated for each mother’s residence using a spatiotemporal interpolation method. Placental 3-NTp levels, adjusted for covariates, increased by 35.0% (95% confidence interval (CI): 13.9, 60.0%) for an interquartile range increment in entire pregnancy PM2.5 exposure. The corresponding estimate for BC exposure was 13.9% (95% CI: -0.21, 29.9%). These results were driven by the first [PM2.5: 29.0% (95% CI: 4.9, 58.6%); BC: 23.6% (95% CI: 4.4, 46.4%)] and second gestational exposure window [PM2.5: 39.3% (95% CI: 12.3, 72.7)]. This link between placental nitrosative stress and exposure to fine particle air pollution during gestation is in line with experimental evidence on cigarette smoke and diesel exhaust exposure. Further research is needed to elucidate potential health consequences later in life through particle-mediated nitrosative stress during fetal life. This work was supported by the European Research Council (Grant ERC-2012-StG 310898) and by the Flemish Scientific Fund (Grants G.073315N, 1516112N and 12D7714N to K.V.).

Epigenetic regulation is hereditable but can be influenced by environmental stimuli, in utero circumstances, and aging. Different layers of epigenetic remodeling including DNA methylation, modifications of histone tails, and noncoding RNAs control the spatial and temporal transcriptomic activity. In addition, the epigenome is involved in sustaining chromosome stability. Genomic DNA isolated from blood cells or other pertinent tissues is being expansively exploited for the discovery of biomarkers of effect and exposure. Technology to measure epigenetic marks on a genomic scale complemented with novel tools for data-analysis have recently been developed and continue to be enhanced. Here, we describe common techniques that are applied for untargeted approaches; and to measure regional modifications and gene-specific aberrations. Alterations in epigenetic marks have been associated with various exposures such as tobacco smoke, air pollution, and metal exposures in population-based studies. On the other hand, deviant DNA methylation is a major epigenetic mechanism of epigenetic silencing in a wide range of human diseases including cancers. Epigenetic modifications might play a prominent role in explaining biological mediation of exposures and their effect on health. This is of particular significance in early life exposures where epigenetic alterations can explain how diseases linked to in utero or childhood conditions occur later in life. We discuss relevant examples of how epigenetic remodeling by environmental stimuli affects several health outcomes in adults and in early life.

The sustainability of cobalt is an important emerging issue because this critical base metal is an essential component of lithium-ion batteries for electric vehicles. More than half of the world’s cobalt mine production comes from the Katanga Copperbelt in DR Congo, with a substantial proportion (estimated at 15–20%) being extracted by artisanal miners. Here we show, in a case study performed in the town of Kolwezi, that people living in a neighbourhood that had been transformed into an artisanal cobalt mine had much higher levels of cobalt in their urine and blood than people living in a nearby control area. The differences were most pronounced for children, in whom we also found evidence of exposure-related oxidative DNA damage. It was already known that industrial mining and processing of metals has led to severe environmental pollution in the region. This field study provides novel and robust empirical evidence that the artisanal extraction of cobalt that prevails in the DR Congo may cause toxic harm to vulnerable communities. This strengthens the conclusion that the currently existing cobalt supply chain is not sustainable. Cobalt is used in rechargeable batteries, and half the world’s supply comes from one district in the Democratic Republic of Congo. This study assesses the sustainability of artisanal mining there, finding much higher levels of cobalt in blood and urine of community members and especially of children.

Introduction Particulate air pollution is probably causally related to increased risk of cardiovascular disease. Plasma homocysteine is an established cardiovascular disease risk factor. Recent studies show that exposure to particulate air pollution is associated with plasma homocysteine levels in adults but no studies on the association between prenatal air pollution and neonatal homocysteine levels exist. Methods In 609 newborns of the ENVIRONAGE (ENVIRonmental influence ON early AGEing) birth cohort, we investigated the association between prenatal particulate matter exposure with a diameter ≤ 2.5 µm (PM2.5) and cord plasma homocysteine levels, and in a subset (n = 490) we studied the interaction with 11 single nucleotide polymorphism (SNPs) in oxidative stress-related genes (CAT, COMT, GSTP1, SOD2, NQO1 and HFE), through multiple linear regression. PM2.5 levels were obtained using a high resolution spatial temporal interpolation method. Homocysteine levels were measured by the homocysteine enzymatic assay on a Roche/Hitachi cobas c system. SNPs were assessed on the Biotrove OpenArray SNP genotyping platform. Results In multivariable-adjusted models, cord plasma homocysteine levels were 8.1% higher (95% CI: 1.9 to 14.3%; p = 0.01) for each 5 µg/m³ increment in average PM2.5 exposure during the entire pregnancy. With regard to pregnancy trimesters, there was only an association in the 2nd trimester: 3.6% (95% CI: 0.9% to 6.4%; p = 0.01). The positive association between PM2.5 in and homocysteine was (borderline) statistically significantly modified by genetic variants in MnSOD (p interaction = 0.02), GSTP1 (p interaction = 0.07) and the sum score of the 3 studied SNPs in the CAT gene (p interaction=0.09), suggesting oxidative stress as an underlying mechanism of action. Conclusions Exposure to particulate air pollution in utero is associated with higher cord blood homocysteine levels, possibly through generating oxidative stress. Increased air pollution-induced homocysteine levels in early life might predispose for cardiovascular and other diseases later in life.

In mammals, a central clock maintains the daily rhythm in accordance with the external environment. At the molecular level, the circadian rhythm is maintained by epigenetic regulation of the Circadian pathway. Here, we tested the role of particulate matter with an aerodynamic diameter ≤ 2.5 μm (PM2.5) exposure during gestational life on human placental Circadian pathway methylation, as an important molecular target for healthy development. In 407 newborns, we quantified placental methylation of CpG sites within the promoter regions of the following genes: CLOCK, BMAL1, NPAS2, CRY1-2 and PER1-3 using bisulfite-PCR-pyrosequencing. Daily PM2.5 exposure levels were estimated for each mother's residence, using a spatiotemporal interpolation model. We applied mixed-effects models to study the methylation status of the Circadian pathway genes and in utero PM2.5 exposure, while adjusting for a priori chosen covariates. In a multi-gene model, placental Circadian pathway methylation was positively and significantly (p

Background: Air pollution exposure during pregnancy has been associated with adverse birth outcomes and health problems later in life. We investigated sex-specific transcriptomic responses to gestational long-and short-term exposure to particulate matter with a diameter < 2.5 mu m ( PM2.5) in order to elucidate potential underlying mechanisms of action. Methods: Whole genome gene expression was investigated in cord blood of 142 mother-newborn pairs that were enrolled in the ENVIRONAGE birth cohort. Daily PM2.5 exposure levels were calculated for each mother's home address using a spatial-temporal interpolation model in combination with a dispersion model to estimate both long- (annual average before delivery) and short- (last month of pregnancy) term exposure. We explored the association between gene expression levels and PM2.5 exposure, and identified modulated pathways by overrepresentation analysis and gene set enrichment analysis. Results: Some processes were altered in both sexes for long-(e.g. DNA damage) or short-term exposure (e.g. olfactory signaling). For long-term exposure in boys neurodevelopment and RhoA pathways were modulated, while in girls defensin expression was down-regulated. For short-term exposure we identified pathways related to synaptic transmission and mitochondrial function (boys) and immune response (girls). Conclusions: This is the first whole genome gene expression study in cord blood to identify sex-specific pathways altered by PM2.5. The identified transcriptome pathways could provide new molecular insights as to the interaction pattern of early life PM2.5 exposure with the biological development of the fetus. This research is funded by the European Research Council (ERC-2012-stG310898) and the Flemish Scientific Fund (FWO, G073315N). Ellen Winckelmans has a PhD. fellowship of Hasselt University (BOF program).

Background: Particulate matter with a diameter ≤ 2.5 μm (PM2.5) affects human fetal development during pregnancy. Oxidative stress is a putative mechanism by which PM2.5 may exert its effects. Leptin (LEP) is an energy-regulating hormone involved in fetal growth and development. Objectives: We investigated in placental tissue whether DNA methylation of the LEP promoter is associated with PM2.5 and whether the oxidative/nitrosative stress biomarker 3-nitrotyrosine (3-NTp) is involved. Methods: LEP DNA methylation status of 361 placentas from the ENVIRONAGE birth cohort was assessed using bisulfite-PCR-pyrosequencing. Placental 3-NTp (n = 313) was determined with an ELISA assay. Daily PM2.5 exposure levels were estimated for each mother’s residence, accounting for residential mobility during pregnancy, using a spatiotemporal interpolation model. Results: After adjustment for a priori chosen covariates, placental LEP methylation was 1.4% lower (95% CI: –2.7, –0.19%) in association with an interquartile range increment (7.5 μg/m3) in second-trimester PM2.5 exposure and 0.43% lower (95% CI: –0.85, –0.02%) in association with a doubling of placental 3-NTp content. Conclusions: LEP methylation status in the placenta was negatively associated with PM2.5 exposure during the second trimester, and with placental 3-NTp, a marker of oxidative/nitrosative stress. Additional research is needed to confirm our findings and to assess whether oxidative/nitrosative stress might contribute to associations between PM2.5 and placental epigenetic events. Potential consequences for health during the neonatal period and later in life warrant further exploration. Citation: Saenen ND, Vrijens K, Janssen BG, Roels HA, Neven KY, Vanden Berghe W, Gyselaers W, Vanpoucke C, Lefebvre W, De Boever P, Nawrot TS. 2017. Lower placental leptin promoter methylation in association with fine particulate matter air pollution during pregnancy and placental nitrosative stress at birth in the ENVIRONAGE cohort. Environ Health Perspect 125:262–268; http://dx.doi.org/10.1289/EHP38

Background: Ultrafine particles (< 100 nm) are ubiquitous present in the air and may contribute to adverse cardiovascular effects. Exposure to air pollutants can alter miRNA expression, which can affect downstream signaling pathways. miRNAs are present both in the intracellular and extracellular environment. In adults, miR-222 and miR-146a were identified as associated with particulate matter exposure. However, there is little evidence of molecular effects of ambient air pollution in children. This study examined whether exposure to fine and ultrafine particulate matter (PM) is associated with changes in the extracellular content of miR-222 and miR-146a of children. Methods: Saliva was collected from 80 children at two different time points, circa 11 weeks apart and stabilized for RNA preservation. The extracellular fraction of saliva was obtained by means of differential centrifugation and ultracentrifugation. Expression levels of miR-222 and miR-146a were profiled by qPCR. We regressed the extracellular miRNA expression against recent exposure to ultrafine and fine particles measured at the school site using mixed models, while accounting for sex, age, BMI, passive smoking, maternal education, hours of television use, time of the day and day of the week. Results: Exposure to ultrafine particles (UFP) at the school site was positively associated with miR-222 expression in the extracellular fraction in saliva. For each IQR increase in particles in the class room (+8504 particles/cm(3)) or playground (+ 28776 particles/cm(3)), miR-222 was, respectively 23.5 % (95 % CI: 3.5 %-41.1 %; p = 0.021) or 29.9 % (95 % CI: 10.6 %-49.1 %; p = 0.0027) higher. No associations were found between miR-146a and recent exposure to fine and ultrafine particles. Conclusions: Our results suggest a possible epigenetic mechanism via which cells respond rapidly to small particles, as exemplified by miR-222 changes in the extracellular fraction of saliva. The COGNAC study is supported by grants from the European Research Council (ERC-2012-StG310898) and the Flemish Scientific Fund (FWO, G073315N). AV has a PhD fellowship from Bijzonder Onderzoeksfonds (BOF) Hasselt University. MK has a PhD fellowship of the Research Foundation Flanders (FWO). EP holds a shared VITO/FWO PhD scholarship. KV has a postdoctoral fellowship of the FWO.

Children's neuropsychological abilities are in a developmental stage. Recent air pollution exposure and neurobehavioralperformance are scarcely studied. In a panel study, we repeatedly administered to each child the followingneurobehavioral tests: Stroop Test (selective attention) and Continuous Performance Test (sustainedattention), Digit Span Forward and Backward Tests (short-term memory), and Digit-Symbol and Pattern ComparisonTests (visual information processing speed). At school, recent inside classroom particulate matter ≤2.5or 10 μm exposure (PM2.5, PM10) was monitored on each examination day. At the child's residence, recent(same day up to 2 days before) and chronic (365 days before examination) exposures to PM2.5, PM10 and blackcarbon (BC) were modeled. Repeated neurobehavioral test performances (n = 894) of the children (n = 310)reflected slower Stroop Test (p = 0.05) and Digit-Symbol Test (p = 0.01) performances with increasing recentinside classroom PM2.5 exposure. An interquartile range (IQR) increment in recent residential outdoor PM2.5 exposurewas associated with an increase in average latency of 0.087 s (SE: ±0.034; p = 0.01) in the Pattern ComparisonTest. Regarding chronic exposure at residence, an IQR increment of PM2.5 exposure was associated withslower performances in the Continuous Performance (9.45 ± 3.47 msec; p = 0.007) and Stroop Tests (59.9 ±26.5 msec; p = 0.02). Similar results were obtained for PM10 exposure. In essence, we showed differential neurobehavioralchanges robustly and adversely associated with recent or chronic ambient exposure to PM air pollutionat residence, i.e., with recent exposure for visual information processing speed (Pattern Comparison Test)and with chronic exposure for sustained and selective attention This work was supported by the Research Foundation-Flanders (FWO, G073315N/G.0873.11.N10) and European Research Council (ERC-2012-StG 310898). Eline B. Provost has a VITO-FWO PhD fellowship. Karen Vrijens has a FWO postdoctoral fellowship (12D7714N).

Introduction Traffic-related air pollution has been shown to induce neurotoxicity in rodents. Several recent epidemiological studies reported negative associations between residential outdoor air pollution and neurobehavioral performance. We investigated in a population of non-smoker adolescents the associations between the urinary concentration of trans, trans-muconic acid (t,t-MA-U), a metabolite of benzene and used as proxy-biomarker of traffic exposure, and two neurobehavioral domains, i.e. sustained attention and short-term memory. Methods In the framework of an environmental health surveillance study in Flanders (Belgium), we examined between 2008 and 2014 grade nine high school students (n=895). We used reaction time, number of omission errors, and number of commission errors in the Continuous Performance Test to evaluate sustained attention, and for the evaluation of short-term memory we used maximum digit span forward and backward of the Digit Span Test. We measured blood lead (PbB) to assess the independent effect of t,t-MA-U on neurobehavioral outcomes. Results This neurobehavioral examination study showed that a ten-fold increase in t,t-MA-U was associated with a 0.14 SD lower sustained attention (95% Confidence Interval: −0.26 to −0.019; p=0.02) and a 0.17 SD diminished short-term memory (95% CI: −0.31 to −0.030; p=0.02). For the same increment in t,t-MA-U, the Continuous Performance Test showed a 12.2 ms higher mean reaction time (95% CI: 4.86–19.5; p=0.001) and 0.51 more numbers of errors of omission (95% CI: 0.057–0.97; p=0.028), while no significant association was found with errors of commission. For the Digit Span Tests, the maximum digit span forward was associated with a 0.20 lower number of digits (95% CI: −0.38 to −0.026; p=0.025) and maximum digit span backward with −0.15 digits (95% CI: −0.32 to 0.022; p=0.088). These associations were independent of PbB, parental education and other important covariates including gender, age, passive smoking, ethnicity, urinary creatinine, time of the day, and examination day of the week. For PbB, an independent association was only found with mean reaction time of the Continuous Performance Test (19.1 ms, 95% CI: 2.43–35.8; p=0.025). Conclusions In adolescents, a ten-fold increase in the concentration of t,t-MA-U, used as a proxy-biomarker for traffic-related exposure, was associated with a significant deficit in sustained attention and short-term memory. The public health implications of this finding cannot be overlooked as the effect-size for these neurobehavioral domains was about 40% of the effect-size of parental education. The study was commissioned and financed by the Ministry of the Flemish Community (Department of Economics, Science and Innovation; Flemish Agency for Care and Health; and Department of Environment, Nature and Energy). This work was further supported by the European Research Council (Grant ERC-2012-StG 310898) and by the Flemish Scientific Fund (FWO) (Grant G.073315N). Michal Kicinski is a Ph.D fellow at the Research Foundation-Flanders (FWO).

Background Developmental processes in the placenta and the fetal brain are shaped by the same biological signals. Recent evidence suggests that adaptive responses of the placenta to the maternal environment may influence central nervous system development. Objectives We studied the association between in utero exposure to fine particle air pollution with a diameter ≤ 2.5 μm (PM2.5) and placental expression of genes implicated in neural development. Methods Expression of 10 target genes in the brain-derived neurotrophic factor (BDNF) signaling pathway were quantified in placental tissue of 90 mother–infant pairs from the ENVIRONAGE birth cohort using quantitative real-time polymerase chain reaction. Trimester-specific PM2.5 exposure levels were estimated for each mother’s home address using a spatiotemporal model. Mixed-effects models were used to evaluate the association between the target genes and PM2.5 exposure measured in different time windows of pregnancy. Results A 5-μg/m3 increase in residential PM2.5 exposure during the first trimester of pregnancy was associated with a 15.9% decrease [95% confidence interval (CI): –28.7, –3.2%, p = 0.015] in expression of placental BDNF at birth. The corresponding estimate for synapsin 1 (SYN1) was a 24.3% decrease (95% CI: –42.8, –5.8%, p = 0.011). Conclusions Placental expression of BDNF and SYN1, two genes implicated in normal neurodevelopmental trajectories, decreased with increasing in utero exposure to PM2.5. Future studies are needed to confirm our findings and evaluate the potential relevance of associations between PM2.5 and placental expression of BDNF and SYN1 on neurodevelopment. We provide the first molecular epidemiological evidence concerning associations between in utero fine particle air pollution exposure and the expression of genes that may influence neurodevelopmental processes. Citation Saenen ND, Plusquin M, Bijnens E, Janssen BG, Gyselaers W, Cox B, Fierens F, Molenberghs G, Penders J, Vrijens K, De Boever P, Nawrot TS. 2015. In utero fine particle air pollution and placental expression of genes in the brain-derived neurotrophic factor signaling pathway: an ENVIRONAGE Birth Cohort Study. Environ Health Perspect 123:834–840; http://dx.doi.org/10.1289/ehp.1408549