Bram G. Janssen

Placental Mitochondrial DNA Content and Particulate Air Pollution during in Utero Life

Background: Studies emphasize the importance of particulate matter (PM) in the formation of reactive oxygen species and inflammation. We hypothesized that these processes can influence mitochondrial function of the placenta and fetus. Objective: We investigated the influence of PM10 exposure during pregnancy on the mitochondrial DNA content (mtDNA content) of the placenta and umbilical cord blood. Methods: DNA was extracted from placental tissue (n = 174) and umbilical cord leukocytes (n = 176). Relative mtDNA copy numbers (i.e., mtDNA content) were determined by real-time polymerase chain reaction. Multiple regression models were used to link mtDNA content and in utero exposure to PM10 over various time windows during pregnancy. Results: In multivariate-adjusted analysis, a 10-µg/m³ increase in PM10 exposure during the last month of pregnancy was associated with a 16.1% decrease [95% confidence interval (CI): –25.2, –6.0%, p = 0.003] in placental mtDNA content. The corresponding effect size for average PM10 exposure during the third trimester was 17.4% (95% CI: –31.8, –0.1%, p = 0.05). Furthermore, we found that each doubling in residential distance to major roads was associated with an increase in placental mtDNA content of 4.0% (95% CI: 0.4, 7.8%, p = 0.03). No association was found between cord blood mtDNA content and PM10 exposure. Conclusions: Prenatal PM10 exposure was associated with placental mitochondrial alterations, which may both reflect and intensify oxidative stress production. The potential health consequences of decreased placental mtDNA content in early life must be further elucidated.

Placental mitochondrial methylation and exposure to airborne particulate matter in the early life environment: An ENVIRONAGE birth cohort study

Most research to date has focused on epigenetic modifications in the nuclear genome, with little attention devoted to mitochondrial DNA (mtDNA). Placental mtDNA content has been shown to respond to environmental exposures that induce oxidative stress, including airborne particulate matter (PM). Damaged or non-functioning mitochondria are specifically degraded through mitophagy, exemplified by lower mtDNA content, and could be primed by epigenetic modifications in the mtDNA. We studied placental mtDNA methylation in the context of the early life exposome. We investigated placental tissue from 381 mother-newborn pairs that were enrolled in the ENVIRONAGE birth cohort. We determined mtDNA methylation by bisulfite-pyrosequencing in 2 regions, i.e., the D-loop control region and 12S rRNA (MT-RNR1), and measured mtDNA content by qPCR. PM2.5 exposure was calculated for each participants home address using a dispersion model. An interquartile range (IQR) increment in PM2.5 exposure over the entire pregnancy was positively associated with mtDNA methylation (MT-RNR1: +0.91%, P = 0.01 and D-loop: +0.21%, P = 0.05) and inversely associated with mtDNA content (relative change of −15.60%, P = 0.001) in placental tissue. mtDNA methylation was estimated to mediate 54% [P = 0.01 (MT-RNR1)] and 27% [P = 0.06 (D-loop)] of the inverse association between PM2.5 exposure and mtDNA content. This study provides new insight into the mechanisms of altered mitochondrial function in the early life environment. Epigenetic modifications in the mitochondrial genome, especially in the MT-RNR1 region, substantially mediate the association between PM2.5 exposure during gestation and placental mtDNA content, which could reflect signs of mitophagy and mitochondrial death.

In Utero Fine Particle Air Pollution and Placental Expression of Genes in the Brain-Derived Neurotrophic Factor Signaling Pathway: An ENVIRONAGE Birth Cohort Study.

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

Biomolecular markers within the core axis of aging and particulate air pollution exposure in the elderly: a cross-sectional study

Background: Telomere length and mitochondrial DNA (mtDNA) content are markers of aging and aging-related diseases. There is inconclusive evidence concerning the mechanistic effects of airborne particulate matter (PM) exposure on biomolecular markers of aging. Objective: The present study examines the association between short- and long-term PM exposure with telomere length and mtDNA content in the elderly and investigates to what extent this association is mediated by expression of genes playing a role in the telomere–mitochondrial axis of aging. Methods: Among 166 nonsmoking elderly participants, we used qPCR to measure telomere length and mtDNA content in leukocytes and RNA from whole blood to measure expression of SIRT1, TP53, PPARGC1A, PPARGC1B, NRF1, and NFE2L2. Associations between PM exposure and markers of aging were estimated using multivariable linear regression models adjusted for sex, age, BMI, socioeconomic status, statin use, past smoking status, white blood cell count, and percentage of neutrophils. Mediation analysis was performed to explore the role of age-related markers between the association of PM exposure and outcome. Annual PM2.5 exposure was calculated for each participant’s home address using a high-resolution spatial–temporal interpolation model. Results: Annual PM2.5 concentrations ranged from 15 to 23 μg/m3. A 5-μg/m3 increment in annual PM2.5 concentration was associated with a relative decrease of 16.8% (95% CI: –26.0%, –7.4%, p = 0.0005) in telomere length and a relative decrease of 25.7% (95% CI: –35.2%, –16.2%, p < 0.0001) in mtDNA content. Assuming causality, results of the mediation analysis indicated that SIRT1 mediated 19.5% and 22.5% of the estimated effect of PM2.5 exposure on telomere length and mtDNA content, respectively. Conclusions: Our findings suggest that the estimated effects of PM2.5 exposure on the telomere–mitochondrial axis of aging may play an important role in chronic health effects of PM2.5. Citation: Pieters N, Janssen BG, Dewitte H, Cox B, Cuypers A, Lefebvre W, Smeets K, Vanpoucke C, Plusquin M, Nawrot TS. 2016. Biomolecular markers within the core axis of aging and particulate air pollution exposure in the elderly: a cross-sectional study. Environ Health Perspect 124:943–950; http://dx.doi.org/10.1289/ehp.1509728

Prenatal Ambient Air Pollution, Placental Mitochondrial DNA Content, and Birth Weight in the INMA (Spain) and ENVIRONAGE (Belgium) Birth Cohorts.

Background: Mitochondria are sensitive to environmental toxicants due to their lack of repair capacity. Changes in mitochondrial DNA (mtDNA) content may represent a biologically relevant intermediate outcome in mechanisms linking air pollution and fetal growth restriction. Objective: We investigated whether placental mtDNA content is a possible mediator of the association between prenatal nitrogen dioxide (NO2) exposure and birth weight. Methods: We used data from two independent European cohorts: INMA (n = 376; Spain) and ENVIRONAGE (n = 550; Belgium). Relative placental mtDNA content was determined as the ratio of two mitochondrial genes (MT-ND1 and MTF3212/R3319) to two control genes (RPLP0 and ACTB). Effect estimates for individual cohorts and the pooled data set were calculated using multiple linear regression and mixed models. We also performed a mediation analysis. Results: Pooled estimates indicated that a 10-μg/m3 increment in average NO2 exposure during pregnancy was associated with a 4.9% decrease in placental mtDNA content (95% CI: –9.3, –0.3%) and a 48-g decrease (95% CI: –87, –9 g) in birth weight. However, the association with birth weight was significant for INMA (–66 g; 95% CI: –111, –23 g) but not for ENVIRONAGE (–20 g; 95% CI: –101, 62 g). Placental mtDNA content was associated with significantly higher mean birth weight (pooled analysis, interquartile range increase: 140 g; 95% CI: 43, 237 g). Mediation analysis estimates, which were derived for the INMA cohort only, suggested that 10% (95% CI: 6.6, 13.0 g) of the association between prenatal NO2 and birth weight was mediated by changes in placental mtDNA content. Conclusion: Our results suggest that mtDNA content can be one of the potential mediators of the association between prenatal air pollution exposure and birth weight. Citation: Clemente DB, Casas M, Vilahur N, Begiristain H, Bustamante M, Carsin AE, Fernández MF, Fierens F, Gyselaers W, Iñiguez C, Janssen BG, Lefebvre W, Llop S, Olea N, Pedersen M, Pieters N, Santa Marina L, Souto A, Tardón A, Vanpoucke C, Vrijheid M, Sunyer J, Nawrot TS. 2016. Prenatal ambient air pollution, placental mitochondrial DNA content, and birth weight in the INMA (Spain) and ENVIRONAGE (Belgium) birth cohorts. Environ Health Perspect 124:659–665; http://dx.doi.org/10.1289/ehp.1408981

Prenatal Air Pollution and Newborns' Predisposition to Accelerated Biological Aging

Importance Telomere length is a marker of biological aging that may provide a cellular memory of exposures to oxidative stress and inflammation. Telomere length at birth has been related to life expectancy. An association between prenatal air pollution exposure and telomere length at birth could provide new insights in the environmental influence on molecular longevity. Objective To assess the association of prenatal exposure to particulate matter (PM) with newborn telomere length as reflected by cord blood and placental telomere length. Design, Setting, and Participants In a prospective birth cohort (ENVIRONAGE [Environmental Influence on Ageing in Early Life]), a total of 730 mother-newborn pairs were recruited in Flanders, Belgium between February 2010 and December 2014, all with a singleton full-term birth (≥37 weeks of gestation). For statistical analysis, participants with full data on both cord blood and placental telomere lengths were included, resulting in a final study sample size of 641. Exposures Maternal residential PM2.5 (particles with an aerodynamic diameter ⩽2.5 &mgr;m) exposure during pregnancy. Main Outcomes and Measures In the newborns, cord blood and placental tissue relative telomere length were measured. Maternal residential PM2.5 exposure during pregnancy was estimated using a high-resolution spatial-temporal interpolation method. In distributed lag models, both cord blood and placental telomere length were associated with average weekly exposures to PM2.5 during pregnancy, allowing the identification of critical sensitive exposure windows. Results In 641 newborns, cord blood and placental telomere length were significantly and inversely associated with PM2.5 exposure during midgestation (weeks 12-25 for cord blood and weeks 15-27 for placenta). A 5-µg/m3 increment in PM2.5 exposure during the entire pregnancy was associated with 8.8% (95% CI, −14.1% to −3.1%) shorter cord blood leukocyte telomeres and 13.2% (95% CI, −19.3% to −6.7%) shorter placental telomere length. These associations were controlled for date of delivery, gestational age, maternal body mass index, maternal age, paternal age, newborn sex, newborn ethnicity, season of delivery, parity, maternal smoking status, maternal educational level, pregnancy complications, and ambient temperature. Conclusions and Relevance Mothers who were exposed to higher levels of PM2.5 gave birth to newborns with shorter telomere length. The observed telomere loss in newborns by prenatal air pollution exposure indicates less buffer for postnatal influences of factors decreasing telomere length during life. Therefore, improvements in air quality may promote molecular longevity from birth onward.

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.

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

Placental Nitrosative Stress and Exposure to Ambient Air Pollution During Gestation: A Population Study

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 the placental 3-NTp levels of 330 mother-newborn pairs enrolled in the Environmental Influence on Ageing in Early Life (ENVIRONAGE) Study, a Belgian birth cohort study (2010-2013). Daily concentrations of particulate matter with an aerodynamic diameter ≤2.5 µm (PM2.5), black carbon (BC), and nitrogen dioxide were interpolated for each mothers 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 each 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 (PM2.5: 39.3% (95% CI: 12.3, 72.7)) gestational exposure windows. 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 experienced later in life through particle-mediated nitrosative stress incurred during fetal life.

Cohort Profile: The ENVIRonmental influence ON early AGEing (ENVIRONAGE): a birth cohort study

The ENVIRONAGE birth cohort is supported by the European Research Council [ERC-2012-StG.310898], and by funds of the Flemish Scientific Research council [FWO, G.0.733.15.N]. Bianca Cox, Janneke Hogervorst and Karen Vrijens have a postdoctoral fellowship from the Research Foundation - Flanders (FWO).

Molecular responses in the telomere-mitochondrial axis of ageing in the elderly: a candidate gene approach.

Experimental evidence shows that telomere shortening induces mitochondrial damage but so far studies in humans are scarce. Here, we investigated the association between leukocyte telomere length (LTL) and mitochondrial DNA (mtDNA) content in elderly and explored possible intermediate mechanisms by determining the gene expression profile of candidate genes in the telomere-mitochondrial axis of ageing. Among 166 non-smoking elderly, LTL, leukocyte mtDNA content and expression of candidate genes: sirtuin1 (SIRT1), tumor protein p53 (TP53), peroxisome proliferator-activated receptor γ-coactivator1α (PGC-1α), peroxisome proliferator-activated receptor γ-coactivator1β (PGC-1β), nuclear respiratory factor 1 (NRF1) and nuclear factor, erythroid 2 like 2 (NRF2), using a quantitave real time polymerase chain assay (qPCR). Statistical mediation analysis was used to study intermediate mechanisms of the telomere-mitochondrial axis of ageing. LTL correlated with leukocyte mtDNA content in our studied elderly (r = 0.23, p = 0.0047). SIRT1 gene expression correlated positively with LTL (r = 0.26, p = 0.0094) and leukocyte mtDNA content (r = 0.43, p < 0.0001). The other studied candidates showed significant correlations in the telomere-mitochondrial interactome but not independent from SIRT1. SIRT1 gene expression was estimated to mediate 40% of the positive association between LTL and leukocyte mtDNA content. The key finding of our study was that SIRT1 expression plays a pivotal role in the telomere-mitochondrial interactome.