Urinary phthalate/DINCH metabolites associations with kisspeptin and reproductive hormones in teenagers: A cross-sectional study from the HBM4EU aligned studies.

BACKGROUND: Exposure to phthalate/DINCH metabolites can induce human reproductive toxicity, however, their endocrine-disrupting mechanisms are not fully elucidated. OBJECTIVE: To investigate the association between concentrations of phthalate/DINCH metabolites, serum kisspeptin, and reproductive hormones among European teenagers from three of the HBM4EU Aligned Studies. METHODS: In 733 Belgian (FLEHS IV study), Slovak (PCB cohort follow-up), and Spanish (BEA study) teenagers, ten phthalate and two DINCH metabolites were measured in urine by high-performance liquid chromatography-tandem mass spectrometry. Serum kisspeptin (kiss54) protein, follicle-stimulating hormone (FSH), total testosterone (TT), estradiol (E2), and sex hormone-binding globulin (SHBG) levels were measured by immunosorbent assays. Free Androgen Index (FAI) was calculated as a proxy of free testosterone. Adjusted sex-stratified linear regression models for individual studies, mixed effect models (LME) accounting for random effects for pooled studies, and g-computation and Bayesian kernel machine regression (BKMR) models for the phthalate/DINCH mixture were performed. RESULTS: The LME suggested that each IQR increase in ln-transformed levels of several phthalates was associated with lower kisspeptin [MnBP: %change (95%CI): -2.8 (-4.2;-0.4); MEHP: -1.4 (-3.4,0.2)] and higher FSH [∑DINP: 11.8 (-0.6;25.1)] levels in females from pooled studies. G-computation showed that the phthalates/DINCH mixture was associated with lower kisspeptin [-4.28 (-8.07;-0.34)] and higher FSH [22.13 (0.5;48.4)] also in females; BKMR showed similar although non-significant pattern. In males, higher phthalates metabolites [MEHP: -12.22 (-21.09;-1.18); oxo-MEHP: -12.73 (-22.34;-1.93)] were associated with lower TT and FAI, although higher DINCH [OH-MINCH: 16.31 (6.23;27.35), cx-MINCH: 16.80 (7.03;27.46), ∑DINCH: 17.37 (7.26;29.74)] were associated with higher TT levels. No mixture associations were found in males. CONCLUSION: We observed sex-specific associations between urinary concentrations of phthalate/DINCH metabolites and the panel of selected effect biomarkers (kisspeptin and reproductive hormones). This suggests that exposure to phthalates would be associated with changes in kisspeptin levels, which would affect the HPG axis and thus influence reproductive health. However, further research is needed, particularly for phthalate replacements such as DINCH.

PFAS and Phthalate/DINCH Exposure in Association with Age at Menarche in Teenagers of the HBM4EU Aligned Studies.

Early puberty has been found to be associated with adverse health outcomes such as metabolic and cardiovascular diseases and hormone-dependent cancers. The decrease in age at menarche observed during the past decades has been linked to an increased exposure to endocrine-disrupting compounds (EDCs). Evidence for the association between PFAS and phthalate exposure and menarche onset, however, is inconsistent. We studied the association between PFAS and phthalate/DINCH exposure and age at menarche using data of 514 teenagers (12 to 18 years) from four aligned studies of the Human Biomonitoring for Europe initiative (HBM4EU): Riksmaten Adolescents 2016-2017 (Sweden), PCB cohort (follow-up; Slovakia), GerES V-sub (Germany), and FLEHS IV (Belgium). PFAS concentrations were measured in blood, and phthalate/DINCH concentrations in urine. We assessed the role of each individual pollutant within the context of the others, by using different multi-pollutant approaches, adjusting for age, age- and sex-standardized body mass index z-score and household educational level. Exposure to di(2-ethylhexyl) phthalate (DEHP), especially mono(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP), was associated with an earlier age at menarche, with estimates per interquartile fold change in 5OH-MEHP ranging from -0.34 to -0.12 years in the different models. Findings from this study indicated associations between age at menarche and some specific EDCs at concentrations detected in the general European population, but due to the study design (menarche onset preceded the chemical measurements), caution is needed in the interpretation of causality.

PFAS association with kisspeptin and sex hormones in teenagers of the HBM4EU aligned studies.

Exposure to Perfluoroalkyl acids (PFAS) can impair human reproductive function, e.g., by delaying or advancing puberty, although their mechanisms of action are not fully understood. We therefore set out to evaluate the relationship between serum PFAS levels, both individually and as a mixture, on the Hypothalamic-Pituitary-Gonadal (HPG) axis by analyzing serum levels of reproductive hormones and also kisspeptin in European teenagers participating in three of the HBM4EU Aligned Studies. For this purpose, PFAS compounds were measured in 733 teenagers from Belgium (FLEHS IV study), Slovakia (PCB cohort follow-up), and Spain (BEA study) by high performance liquid chromatography-tandem mass spectrometry (HPLC/MS) in laboratories under the HBM4EU quality assurance quality control (QA/QC) program. In the same serum samples, kisspeptin 54 (kiss-54) protein, follicle-stimulating hormone (FSH), total testosterone (TT), estradiol (E2), and sex hormone-binding globulin (SHBG) levels were also measured using immunosorbent assays. Sex-stratified single pollutant linear regression models for separate studies, mixed single pollutant models accounting for random effects for pooled studies, and g-computation and Bayesian kernel machine regression (BKMR) models for the mixture of the three most available (PFNA, PFOA, and PFOS) were fit. PFAS associations with reproductive markers differed according to sex. Each natural log-unit increase of PFOA, PFNA, and PFOS were associated with higher TT [18.41 (6.18; 32.31), 15.60 (7.25; 24.61), 14.68 (6.18; 24.61), respectively] in girls, in the pooled analysis (all studies together). In males, G-computation showed that PFAS mixture was associated with lower FSH levels [-10.51 (-18.81;-1.36)]. The BKMR showed the same patterns observed in G-computation, including a significant increase on male Kiss-54 and SHBG levels. Overall, effect biomarkers may enhance the current epidemiological knowledge regarding the adverse effect of PFAS in human HPG axis, although further research is warranted.

Calibration of the comet assay using ionising radiation.

Several trials have attempted to identify sources of inter-laboratory variability in comet assay results, aiming at achieving more equal responses. Ionising radiation induces a defined level of DNA single-strand breaks (per dose/base pairs) and is used as a reference when comparing comet results but relies on accurately determined radiation doses. In this ring test we studied the significance of dose calibrations and comet assay protocol differences, with the object of identifying causes of variability and how to deal with them. Eight participating laboratories, using either x-ray or gamma radiation units, measured dose rates using alanine pellet dosimeters that were subsequently sent to a specialised laboratory for analysis. We found substantial deviations between calibrated and nominal (uncalibrated) dose rates, with up to 46% difference comparing highest and lowest values. Three additional dosimetry systems were employed in some laboratories: thermoluminescence detectors and two aqueous chemical dosimeters. Fricke's and Benzoic Acid dosimetry solutions gave reliable quantitative dose estimations using local equipment. Mononuclear cells from fresh human blood or mammalian cell lines were irradiated locally with calibrated (alanine) radiation doses and analysed for DNA damage using a standardised comet assay protocol and a lab-specific protocol. The dose response of eight laboratories, calculated against calibrated radiation doses, was linear with slope variance CV= 29% with the lab-specific protocol, reduced to CV= 16% with the standard protocol. Variation between laboratories indicate post-irradiation repair differences. Intra-laboratory variation was very low judging from the dose response of 8 donors (CV=4%). Electrophoresis conditions were different in the lab-specific protocols explaining some dose response variations which were reduced by systematic corrections for electrophoresis conditions. The study shows that comet assay data obtained in different laboratories can be compared quantitatively using calibrated radiation doses and that systematic corrections for electrophoresis conditions are useful.

Validation of a food-frequency questionnaire to assess methyl-group donor intake in preschoolers.

A good assessment of dietary methyl-group donor intake (folate, choline, betaine, methionine) is needed to investigate the effect of methyl-group donor intake on children's health. The aim is to develop and validate a food-frequency questionnaire (FFQ) to estimate the daily intake of methyl-group donors in preschoolers. For the relative validity and reproducibility of the FFQ, a 7-day estimated dietary record (7d EDR) and repeated measurements 6 weeks apart (n = 77) were used respectively. For the validity (n = 75), a moderate ranking ability was obtained (de-attenuated Pearson correlation = 0.43-0.70; weighted ƙ 0.10-0.40), but the FFQ tends to overestimate the real intake. Cross-classification analysis showed that 38% (choline) and 28% (betaine) of the subjects were misclassified. The FFQ shows a moderate to good reproducibility, intra-class correlation coefficients range between 0.67 (betaine) and 0.76 (choline) with a maximal misclassification of 6.5% for betaine (weighted ƙ 0.37-0.69) (n = 77). CONCLUSION: The FFQ is a tool with a moderate validity and reproducibility to estimate the consumption of dietary methyl-group donors in preschoolers. This tool might be used in future research epidemiological studies including preschoolers. WHAT IS KNOWN: • Poor dietary habits during childhood have been associated with chronic disease in adulthood. • Methyl-group donors are important for DNA methylation, which is the underlying mechanism of disease development. WHAT IS NEW: • For the first time, a food-frequency questionnaire that can assess the intake of methione, folate, choline, and betaine has been developed and validated for the use in preschoolers.

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

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

The hCOMET project: International database comparison of results with the comet assay in human biomonitoring. Baseline frequency of DNA damage and effect of main confounders.

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.

Exposure levels, determinants and risk assessment of organophosphate flame retardants and plasticizers in adolescents (14-15 years) from the Flemish Environment and Health Study.

The ubiquitous use of organophosphate flame retardants and plasticizers (PFRs) in a variety of consumer products has led to widespread human exposure. Since certain PFRs are developmental and carcinogenic toxicants, detailed exposure assessments are essential to investigate the risk associated with environmental exposure levels. However, such data are still lacking for European countries. In this study, concentrations of thirteen PFR metabolites were measured in urine samples from 600 adolescents from Flanders, Belgium. 1-Hydroxy-2-propyl bis(1-chloro-2-propyl) phosphate (BCIPHIPP), diphenyl phosphate (DPHP), bis(1,3-dichloro-isopropyl) phosphate (BDCIPP), 2-hydroxyethyl bis(2-butoxyethyl) phosphate (BBOEHEP), 2-ethylhexyl phenyl phosphate (EHPHP) and 2-ethyl-5-hydroxyhexyl diphenyl phosphate (5-HO-EHDPHP) were frequently detected (>83%) in all participants. Comparisons with study populations from outside the EU showed that urinary levels of DPHP, BDCIPP and BCIPHIPP were generally within the same range. Only exposure to 2-ethylhexyl diphenyl phosphate (EHDPHP) was presumably higher in Flemish adolescents. However, determinants analysis through multivariate regression analyses did not reveal significant predictors that may explain this finding. Significantly higher levels of BDCIPP were observed in participants with new decorations at home, while adolescents with highly educated parents had higher levels of BBOEHEP and BDCIPP. Furthermore, multiple PFR metabolite concentrations followed a seasonal pattern. Estimated daily intakes (EDIs) were calculated from the internal dose by including fractions of urinary excretion (FUE) estimated in in vitro metabolism studies. EDIs ranged from 6.3 ng/kg bw/day for TBOEP to 567.7 ng/kg bw/day for EHDPHP, which were well below the available oral reference doses for all investigated PFRs. This suggests that the associated risk is low at present. This is the first report on internal exposure to seven commonly used PFRs in a European population.

Real-time versus thermal desorption selected ion flow tube mass spectrometry for quantification of breath volatiles.

RATIONALE: Selected ion flow tube mass spectrometry (SIFT-MS) is versatile, rapidly provides result output and determines a wide range of volatiles, making it suitable for biomedical applications. When direct sampling into the SIFT-MS instrument is impractical, combining thermal desorption (TD) and SIFT-MS might offer a solution as it allows sample storage on sorbent tubes for later analysis. This work compares off-line TD SIFT-MS and real-time SIFT-MS for the quantification of selected breath volatiles. METHODS: Ten healthy non-smoking individuals provided 60 breath samples per method. For off-line analysis, breath was collected onto sorbent tubes via a breath sampler provided with filtered inspiratory air. After TD, samples were re-collected in Tedlar bags which were then connected to the SIFT-MS instrument. For real-time analysis, breath was sampled directly into the instrument. In both cases the analytical method included a total of 155 product ions, and 14 selected volatiles were quantified. The agreement between the methods was assessed using Pearson correlation coefficients and Bland-Altman plots. RESULTS: Overall, correlations between real-time and off-line analysis were moderate to very strong (r = 0.43-0.92) depending on the volatile of interest, except for 2,3-butanedione and styrene. The difference between real-time and off-line measured breath concentrations (average bias) ranged between -14.57 and 20.48 ppbv. For acetone and isoprene, it was 251.53 and 31.9 ppbv, respectively. CONCLUSIONS: Real-time SIFT-MS and off-line TD SIFT-MS for quantification of selected breath volatiles did not show optimal agreement. Analyzing a multitude of analytes in breath via direct exhalation into a SIFT-MS instrument for real-time analysis is challenging. On the other hand, off-line analysis using a breath collection device also has its issues such as possible sample losses due to selective absorption depending on the sorbent used or during desorption and transfer to the instrument. Despite these drawbacks, both methods were moderately well correlated.

Determinants of exposure levels of bisphenols in flemish adolescents.

The broadly used industrial chemical bisphenol A (BPA), applied in numerous consumer products, has been under scrutiny in the past 20 years due to its widespread detection in humans and the environment and potential detrimental effects on human health. Following implemented restrictions and phase-out initiatives, BPA is replaced by alternative bisphenols, which have not received the same amount of research attention. As a part of the fourth cycle of the Flemish Environment and Health Study (FLEHS IV, 2016-2020), we monitored the internal exposure to six bisphenols in urine samples of 423 adolescents (14-15 years old) from Flanders, Belgium. All measured bisphenols were detected in the study population, with BPA and its alternatives bisphenol F (BPF) and bisphenol S (BPS) showing detection frequencies > 50%. The reference values show that exposure to these compounds is extensive. However, the urinary BPA level decreased significantly in Flemish adolescents compared to a previous cycle of the FLEHS (2008-2009). This suggests that the replacement of BPA with its analogues is ongoing. Concentrations of bisphenols measured in the Flemish adolescents were generally in the same order of magnitude compared to recent studies worldwide. Multiple regression models were used to identify determinants of exposure based on information on demographic and lifestyle characteristics of participants, acquired through questionnaires. Some significant determinants could be identified: sex, season, smoking behavior, educational level of the parents, recent consumption of certain foods and use of certain products were found to be significantly associated with levels of bisphenols. Preliminary risk assessment showed that none of the estimated daily intakes (EDIs) of BPA exceeded the tolerable daily intake, even in a high exposure scenario. For alternative bisphenols, no health-based guidance values are available, but in line with the measured urinary levels, their EDIs were lower than that of BPA. This study is, to the best of our knowledge, the first to determine internal exposure levels of other bisphenols than BPA in a European adolescent population.

Application of the comet assay in human biomonitoring: An hCOMET perspective.

The comet assay is a well-accepted biomonitoring tool to examine the effect of dietary, lifestyle, environmental and occupational exposure on levels of DNA damage in human cells. With such a wide range of determinants for DNA damage levels, it becomes challenging to deal with confounding and certain factors are inter-related (e.g. poor nutritional intake may correlate with smoking status). This review describes the effect of intrinsic (i.e. sex, age, tobacco smoking, occupational exposure and obesity) and extrinsic (season, environmental exposures, diet, physical activity and alcohol consumption) factors on the level of DNA damage measured by the standard or enzyme-modified comet assay. Although each factor influences at least one comet assay endpoint, the collective evidence does not indicate single factors have a large impact. Thus, controlling for confounding may be necessary in a biomonitoring study, but none of the factors is strong enough to be regarded a priori as a confounder. Controlling for confounding in the comet assay requires a case-by-case approach. Inter-laboratory variation in levels of DNA damage and to some extent also reproducibility in biomonitoring studies are issues that have haunted the users of the comet assay for years. Procedures to collect specimens, and their storage, are not standardized. Likewise, statistical issues related to both sample-size calculation (before sampling of specimens) and statistical analysis of the results vary between studies. This review gives guidance to statistical analysis of the typically complex exposure, co-variate, and effect relationships in human biomonitoring studies.

Prenatal exposure to endocrine disrupting chemicals and risk of being born small for gestational age: Pooled analysis of seven European birth cohorts.

BACKGROUND AND AIMS: There is evidence that endocrine disrupting chemicals (EDCs) have developmental effects at environmental concentrations. We investigated whether some EDCs are associated with the adverse birth outcome Small for Gestational Age (SGA). METHODS: We used PCB 153, p,p'-DDE, HCB, PFOS and PFOA measured in maternal, cord blood or breast milk samples of 5446 mother-child pairs (subset of 693 for the perfluorinated compounds) from seven European birth cohorts (1997-2012). SGA infants were those with birth weight below the 10th percentile for the norms defined by gestational age, country and infant's sex. We modelled the association between measured or estimated cord serum EDC concentrations and SGA using multiple logistic regression analyses. We explored effect modification by child's sex and maternal smoking during pregnancy. RESULTS: Among the 5446 newborns, 570 (10.5%) were SGA. An interquartile range (IQR) increase in PCB 153 was associated with a modestly increased risk of SGA (odds ratio (OR) of 1.05 [95% CI: 1.04-1.07]) that was stronger in girls (OR of 1.09 [95% CI: 1.04-1.14]) than in boys (OR of 1.03 [95% CI: 1.03-1.04]) (p-interaction = 0.025). For HCB, we found a modestly increased odds of SGA in girls (OR of 1.04 [95% CI: 1.01-1.07] per IQR increase), and an inverse association in boys (OR of 0.90 [95% CI: 0.85-0.95]) (p-interaction = 0.0003). Assessment of the HCB-sex-smoking interaction suggested that the increased odds of SGA associated with HCB exposure was only in girls of smoking mothers (OR of 1.18 [95% CI: 1.11-1.25]) (p-interaction = 0.055). Higher concentrations of PFOA were associated with greater risk of SGA (OR of 1.64 [95% CI: 0.97-2.76]). Elevated PFOS levels were associated with increased odds of SGA in newborns of mothers who smoked during pregnancy (OR of 1.63 [95% CI: 1.02-2.59]), while an inverse association was found in those of non-smoking mothers (OR of 0.66 [95% CI: 0.61-0.72]) (p-interaction = 0.0004). No significant associations were found for p,p'-DDE. CONCLUSIONS: Prenatal environmental exposure to organochlorine and perfluorinated compounds with endocrine disrupting properties may contribute to the prevalence of SGA. We found indication of effect modification by child's sex and smoking during pregnancy. The direction of the associations differed by chemical and these effect modifiers, suggesting diverse mechanisms of action and biological pathways.

Planarians Customize Their Stem Cell Responses Following Genotoxic Stress as a Function of Exposure Time and Regenerative State.

Aiming to in vivo characterize the responses of pluripotent stem cells and regenerative tissues to carcinogenic stress, we employed the highly regenerative organism Schmidtea mediterranea. Its broad regenerative capacities are attributable to a large pool of pluripotent stem cells, which are considered key players in the lower vulnerability toward chemically induced carcinogenesis observed in regenerative organisms. Schmidtea mediterranea is, therefore, an ideal model to study pluripotent stem cell responses with stem cells residing in their natural environment. Including microenvironmental alterations is important, as the surrounding niche influences the onset of oncogenic events. Both short- (3 days) and long-term (17 days) exposures to the genotoxic carcinogen methyl methanesulfonate (50 µM) were evaluated during homeostasis and animal regeneration, two situations that render altered cellular niches. In both cases, MMS-induced DNA damage was observed, which provoked a decrease in proliferation on the short term. The outcome of DNA damage responses following long-term exposure differed between homeostatic and regenerating animals. During regeneration, DNA repair systems were more easily activated than in animals in homeostasis, where apoptosis was an important outcome. Knockdown experiments confirmed the importance of DNA repair systems during carcinogenic exposure in regenerating animals as knockdown of rad51 induced a stem cell-depleted phenotype, after regeneration was completed.

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

BACKGROUND: Maternal nutrition during pregnancy and infant nutrition in the early postnatal period (lactation) are critically involved in the development and health of the newborn infant. The Maternal Nutrition and Offspring's Epigenome (MANOE) study was set up to assess the effect of maternal methyl-group donor intake (choline, betaine, folate, methionine) on infant DNA methylation. Maternal intake of dietary methyl-group donors was assessed using a food-frequency questionnaire (FFQ). Before and during pregnancy, we evaluated maternal methyl-group donor intake through diet and supplementation (folic acid) in relation to gene-specific (IGF2 DMR, DNMT1, LEP, RXRA) buccal epithelial cell DNA methylation in 6 months old infants (n = 114) via pyrosequencing. In the early postnatal period, we determined the effect of maternal choline intake during lactation (in mothers who breast-fed for at least 3 months) on gene-specific buccal DNA methylation (n = 65). RESULTS: Maternal dietary and supplemental intake of methyl-group donors (folate, betaine, folic acid), only in the periconception period, was associated with buccal cell DNA methylation in genes related to growth (IGF2 DMR), metabolism (RXRA), and appetite control (LEP). A negative association was found between maternal folate and folic acid intake before pregnancy and infant LEP (slope = -1.233, 95% CI -2.342; -0.125, p = 0.0298) and IGF2 DMR methylation (slope = -0.706, 95% CI -1.242; -0.107, p = 0.0101), respectively. Positive associations were observed for maternal betaine (slope = 0.875, 95% CI 0.118; 1.633, p = 0.0241) and folate (slope = 0.685, 95% CI 0.245; 1.125, p = 0.0027) intake before pregnancy and RXRA methylation. Buccal DNMT1 methylation in the infant was negatively associated with maternal methyl-group donor intake in the first and second trimester of pregnancy and negatively in the third trimester. We found no clear association between maternal choline intake during lactation and buccal infant DNA methylation. CONCLUSIONS: This study suggests that maternal dietary and supplemental intake of methyl-group donors, especially in the periconception period, can influence infant's buccal DNA methylation in genes related to metabolism, growth, appetite regulation, and maintenance of DNA methylation reactions.

Environmental exposure to human carcinogens in teenagers and the association with DNA damage.

BACKGROUND: We investigated whether human environmental exposure to chemicals that are labeled as (potential) carcinogens leads to increased (oxidative) damage to DNA in adolescents. MATERIAL AND METHODS: Six hundred 14-15-year-old youngsters were recruited all over Flanders (Belgium) and in two areas with important industrial activities. DNA damage was assessed by alkaline and formamidopyrimidine DNA glycosylase (Fpg) modified comet assays in peripheral blood cells and analysis of urinary 8-hydroxydeoxyguanosine (8-OHdG) levels. Personal exposure to potentially carcinogenic compounds was measured in urine, namely: chromium, cadmium, nickel, 1-hydroxypyrene as a proxy for exposure to other carcinogenic polycyclic aromatic hydrocarbons (PAHs), t,t-muconic acid as a metabolite of benzene, 2,5-dichlorophenol (2,5-DCP), organophosphate pesticide metabolites, and di(2-ethylhexyl) phthalate (DEHP) metabolites. In blood, arsenic, polychlorinated biphenyl (PCB) congeners 118 and 156, hexachlorobenzene (HCB), dichlorodiphenyltrichloroethane (DDT) and perfluorooctanoic acid (PFOA) were analyzed. Levels of methylmercury (MeHg) were measured in hair. Multiple linear regression models were used to establish exposure-response relationships. RESULTS: Biomarkers of exposure to PAHs and urinary chromium were associated with higher levels of both 8-OHdG in urine and DNA damage detected by the alkaline comet assay. Concentrations of 8-OHdG in urine increased in relation with increasing concentrations of urinary t,t-muconic acid, cadmium, nickel, 2,5-DCP, and DEHP metabolites. Increased concentrations of PFOA in blood were associated with higher levels of DNA damage measured by the alkaline comet assay, whereas DDT was associated in the same direction with the Fpg-modified comet assay. Inverse associations were observed between blood arsenic, hair MeHg, PCB 156 and HCB, and urinary 8-OHdG. The latter exposure biomarkers were also associated with higher fish intake. Urinary nickel and t,t-muconic acid were inversely associated with the alkaline comet assay. CONCLUSION: This cross-sectional study found associations between current environmental exposure to (potential) human carcinogens in 14-15-year-old Flemish adolescents and short-term (oxidative) damage to DNA. Prospective follow-up will be required to investigate whether long-term effects may occur due to complex environmental exposures.

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

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

Identification of PM10 characteristics involved in cellular responses in human bronchial epithelial cells (Beas-2B).

Notwithstanding evidence is present that physicochemical characteristics of ambient particles attribute to adverse health effects, there is still some lack of understanding in this complex relationship. At this moment it is not clear which properties (such as particle size, chemical composition) or sources of the particles are most relevant for health effects. This study investigates the in vitro toxicity of PM10 in relation to PM chemical composition, black carbon (BC), endotoxin content and oxidative potential (OP). In 2013-2014 PM10 was sampled (24h sampling, 108 sampling days) in ambient air at three sites in Flanders (Belgium) with different pollution characteristics: an urban traffic site (Borgerhout), an industrial area (Zelzate) and a rural background location (Houtem). To characterize the toxic potential of PM10, airway epithelial cells (Beas-2B cells) have been exposed to particles in vitro. Different endpoints were studied including cell damage and death (cell viability) using the Neutral red Uptake assay, the production of pro-inflammatory molecules by interleukin 8 (IL-8) induction and DNA-damaging activity using the FPG-modified Comet assay. The endotoxin levels in the collected samples were analysed and the capacity of PM10 particles to produce reactive oxygen species (OP) was evaluated by electron paramagnetic resonance (EPR) spectroscopy. Chemical characteristics of PM10 (BC, As, Cd, Cr, Cu, Mn, Ni, Pb, Zn) and meteorological conditions were recorded on the sampling days. PM10 particles exhibited dose-dependent cytotoxicity in Beas-2B cells and were found to significantly induce the release of IL-8 in samples from the three locations. Oxidatively damaged DNA was observed in exposed Beas-2B cells. Endotoxin levels above the detection limit were detected in half of the samples. OP was measurable in all samples. Associations between PM10 characteristics and biological effects of PM10 were assessed by single and multiple regression analyses. The reduction in cell viability was significantly correlated with BC, Cd and Pb. The induction of IL-8 in Beas-2B cells was significantly associated with Cu, Ni and Zn and endotoxin. Endotoxin levels explained 33% of the variance in IL-8 induction. A significant interaction between ambient temperature and endotoxin on the pro-inflammatory activity was seen. No association was found between OP and the cellular responses. This study supports the hypothesis that, on an equal mass basis, PM10 induced biological effects differ due to differences in PM10 characteristics. Metals (Cd, Cu, Ni and Zn), BC, and endotoxin were among the main determinants for the observed biological responses.

12 Weeks of Combined Endurance and Resistance Training Reduces Innate Markers of Inflammation in a Randomized Controlled Clinical Trial in Patients with Multiple Sclerosis.

Previously, we reported that patients with multiple sclerosis (MS) demonstrate improved muscle strength, exercise tolerance, and lean tissue mass following a combined endurance and resistance exercise program. However, the effect of exercise on the underlying disease pathogenesis remains elusive. Since recent evidence supports a crucial role of dendritic cells (DC) in the pathogenesis of MS, we investigated the effect of a 12-week combined exercise program in MS patients on the number and function of DC. We demonstrate an increased number of plasmacytoid DC (pDC) following the exercise program. These pDC display an activated phenotype, as evidenced by increased numbers of circulating CD62L(+) and CD80(+) pDC. Interestingly, the number of CD80(+) pDC positively correlates with the presence of IL-10-producing regulatory type 1 cells (Tr1), an important cell type for maintaining peripheral tolerance to self-antigens. In addition, decreased production of the inflammatory mediators, TNF-α and MMP-9, upon Toll-like receptor (TLR) stimulation was found at the end of the exercise program. Overall, our findings suggest that the 12-week exercise program reduces the secretion of inflammatory mediators upon TLR stimulation and promotes the immunoregulatory function of circulating pDC, suggestive for a favorable impact of exercise on the underlying immunopathogenesis of MS.

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

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

Reproducibility and validity of an FFQ to assess usual intake of methyl-group donors.

OBJECTIVE: To develop and validate a novel FFQ to assess the daily intake of four methyl-group donors (methionine, choline, betaine and folate). DESIGN: The relative validity of the FFQ was assessed by comparison with 7 d estimated diet records (n 80) and its reproducibility was evaluated by repeated administrations 6 weeks apart (n 92). Paired Student t tests were used to compare group means and de-attenuated intra-class correlations to investigate the ability of the FFQ to rank individuals according to their methyl-group donor intake. De-attenuated intra-class correlation coefficients were calculated between the test and reference method for methionine, choline, betaine, folate and the sum of methyl-group donors. The weighted kappa (κ w) was calculated as a measure of tertile agreement. SETTING: Flanders, Belgium. SUBJECTS: The FFQ was validated among Flemish women of reproductive age (18-35 years). RESULTS: The questionnaire had an acceptable ranking ability (r=0·32-0·68; κ w=0·10-0·35), but overestimated the daily intake of folate (280·6 µg v. 212·0 µg) and betaine (179·1 mg v. 147·0 mg) compared with the 7 d estimated diet record. Cross-classification analysis indicated that 20 % (choline) of the participants were grossly misclassified in the validation study. The correlation between repeated administrations was good (r=0·62-0·83) with a maximal misclassification of 7 % for betaine (κ w=0·44-0·66). CONCLUSIONS: These results indicate that this newly developed FFQ is a reliable instrument with acceptable validity for ranking individuals according to methyl-group donor intake (except for a poor agreement for choline (κ w=0·10) and a fair ranking ability for betaine (r=0·32)) in Flemish women of reproductive age.