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Per-and polyfluoroalkyl substances (PFAS) are widespread pollutants that may impact youth adiposity patterns. We investigated cross-sectional associations between PFAS and body mass index (BMI) in teenagers/adolescents across nine European countries within the Human Biomonitoring for Europe (HBM4EU) initiative. We used data from 1957 teenagers (12-18 yrs) that were part of the HBM4EU aligned studies, consisting of nine HBM studies (NEBII, Norway; Riksmaten Adolescents 2016-17, Sweden; PCB cohort (follow-up), Slovakia; SLO CRP, Slovenia; CROME, Greece; BEA, Spain; ESTEBAN, France; FLEHS IV, Belgium; GerES V-sub, Germany). Twelve PFAS were measured in blood, whilst weight and height were measured by field nurse/physician or self-reported in ques-tionnaires. We assessed associations between PFAS and age-and sex-adjusted BMI z-scores using linear and logistic regression adjusted for potential confounders. Random-effects meta-analysis and mixed effects models were used to pool studies. We assessed mixture effects using molar sums of exposure biomarkers with toxicological/structural similarities and quantile g-computation. In all studies, the highest concentrations of PFAS were PFOS (medians ranging from 1.34 to 2.79 mu g/L). There was a tendency for negative associations with BMI z-scores for all PFAS (except for PFHxS and PFHpS), which was borderline significant for the molar sum of [PFOA and PFNA] and significant for single PFOA [beta-coefficient (95% CI) per interquartile range fold change =-0.06 (-0.17, 0.00) and-0.08 (-0.15,-0.01), respectively]. Mixture assessment indicated similar negative associations of the total mixture of [PFOA, PFNA, PFHxS and PFOS] with BMI z-score, but not all compounds showed associations in the same direction: whilst [PFOA, PFNA and PFOS] were negatively associated, [PFHxS] associated positively with BMI z-score. Our results indicated a tendency for associations of relatively low PFAS concentrations with lower BMI in European teenagers. More prospective research is needed to investigate this potential relationship and its implications for health later in life., European Commission 733032, Swedish Research Council European Commission 2017-00822

Human biomonitoring (HBM) studies have highlighted widespread daily exposure to environmental chemicals. Some of these are suspected to contribute to adverse health outcomes such as reproductive, neurological, and metabolic disorders, among other developmental and chronic impairments. One of the objectives of the H2020 European Human Biomonitoring Initiative (HBM4EU) was the development of informative effect biomarkers for application in a more systematic and harmonized way in large-scale European HBM studies. The inclusion of effect biomarkers would complement exposure data with mechanistically-based information on early and late adverse effects. For this purpose, a stepwise strategy was developed to identify and implement a panel of vali- dated effect biomarkers in European HBM studies. This work offers an overview of the complete procedure followed, from comprehensive literature search strategies, selection of criteria for effect biomarkers and their classification and prioritization, based on toxi- cological data and adverse outcomes, to pilot studies for their analytical, physiological, and epidemiological validation. We present the example of one study that demonstrated the mediating role of the effect biomarker status of brain-derived neurotrophic factor BDNF in the longitudinal association between infant bisphenol A (BPA) exposure and behavioral function in adolescence. A panel of effect biomarkers has been implemented in the HBM4EU Aligned Studies as main outcomes, including traditional oxidative stress, reproductive, and thyroid hormone biomarkers. Novel biomarkers of effect, such as DNA methylation status of BDNF and kisspeptin (KISS) genes were also evaluated as molecular markers of neurological and reproductive health, respectively. A panel of effect biomarkers has also been applied in HBM4EU occupational studies, such as micronucleus analysis in lymphocytes and reticulocytes, whole blood comet assay, and malondialdehyde, 8-oxo-2′ -deoxyguanosine and untargeted metabolomic profile in urine, to investigate, for example, biological changes in response to hexavalent chromium Cr(VI) exposure. The use of effect biomarkers in HBM4EU has demonstrated their ability to detect early biological effects of chemical exposure and to identify subgroups that are at higher risk. The roadmap developed in HBM4EU con- firms the utility of effect biomarkers, and support one of the main objectives of HBM research, which is to link exposure biomarkers to mechanistically validated effect and susceptibility biomarkers in order to better un- derstand the public health implications of human exposure to environmental chemicals., European Union’s Horizon 2020 research and innovation program projects HBM4EU 733032

While human regulatory risk assessment (RA) still largely relies on animal studies, new approach methodologies (NAMs) based on in vitro, in silico or non-mammalian alternative models are increasingly used to evaluate chemical hazards. Moreover, human epidemiological studies with biomarkers of effect (BoE) also play an invaluable role in identifying health effects associated with chemical exposures. To move towards the next generation risk assessment (NGRA), it is therefore crucial to establish bridges between NAMs and standard approaches, and to establish processes for increasing mechanistically-based biological plausibility in human studies. The Adverse Outcome Pathway (AOP) framework constitutes an important tool to address these needs but, despite a significant increase in knowledge and awareness, the use of AOPs in chemical RA remains limited. The objective of this paper is to address issues related to using AOPs in a regulatory context from various perspectives as it was discussed in a workshop organized within the European Union partnerships HBM4EU and PARC in spring 2022. The paper presents examples where the AOP framework has been proven useful for the human RA process, particularly in hazard prioritization and characterization, in integrated approaches to testing and assessment (IATA), and in the identification and validation of BoE in epidemiological studies. Nevertheless, several limitations were identified that hinder the optimal usability and acceptance of AOPs by the regulatory community including the lack of quantitative information on response-response relationships and of efficient ways to map chemical data (exposure and toxicity) onto AOPs. The paper summarizes suggestions, ongoing initiatives and third-party tools that may help to overcome these obstacles and thus assure better implementation of AOPs in the NGRA., European Commission 733032 857560 101057014, Ministry of Education, Youth and Sports by the RECETOX Research Infrastructure LM2018121, OP RDE project CETOCOEN Excellence CZ.02.1.01/0.0/0.0/17_043/0009632, Japan Agency for Medical Research and Development (AMED) JP21mk0101216 JP22mk0101216, Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT), Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research (KAKENHI) 21K12133

Objective: To assess the genetic and epigenetic effects promoted by Bisphenol A(BPA) exposure in adolescent males from the Spanish INMA-Granada birth cohort, as well as in human cells. Methods: DNA methylation was analysed using MEDIP. Repeat number variation in genomic DNA was evaluated, along with the analysis of H3K4me3 by using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). All experiments were performed with material extracted from whole blood of adolescents from INMA. The epidemiological study was complemented by in vitro assessments of human (HeLa) cells exposed to BPA, specifically, immunofluorescence evaluation of histone modification levels, gene expression analysis and ChIP‒qPCR analysis. Results: Adolescents in the high urinary BPA group presented higher genetic instability of Satellite A (SATA) repetitive region compared to those in the low BPA group. We also observed decreased DNA methylation at the promoters of the imprinted genes H19, KCNQ1, and IGF2; at LINE1 retroelements; and at the ARID2, EGFR1 and ESRRA genes. Genome-wide sequencing revealed increased H3K4me3 occupancy at the promoters of genes encoding histone acetyltransferases, telomeric DNA binding factors and DNA repair genes. These results were supported by studying HeLa cells exposed to 10 nMBPA in vitro. Exposure of cells to BPA caused a global increase in histone H4 acetylation and a decrease in H3K9me3 levels. In exposed cells, changes in the expression of genes encoding DNA repair factors (ATM, ARID2) were observed, and the expression of several genesencoding telomeric DNA binding factors (SMG7, TERT, TEN1, UPF1, ZBTB48) increased. Moreover, increased binding of ESR1 to KAT5, KMT2E and TERF2IP promoters and decreased ESR1 binding at the RARA promoter were observed. Conclusion: Genome-wide analysis of histone trimethylation and BPA exposure in the in adolescents from the INMA cohort revealed a global impact of BPA on the expression of genes encoding telomeric binding proteins and histone acetyltransferase factors, which showed parallels with HeLa cells exposed to a human-relevant dose.

Aluminum (Al), antimony (Sb), and lithium (Li) are relatively common toxic metal(oid)s that can be transferred into breast milk and potentially to the nursing infant. This study assessed concentrations of Al, Sb, and Li in breast milk samples collected from donor mothers and explored the predictors of these concentrations. Two hundred forty-two pooled breast milk samples were collected at different times post-partum from 83 donors in Spain (2015-2018) and analyzed for Al, Sb, and Li concentrations. Mixed-effect linear regression was used to investigate the association of breast milk concentrations of these elements with the sociodemographic profile of the women, their dietary habits and utilization of personal care products (PCPs), the post-partum interval, and the nutritional characteristics of milk samples, among other factors. Al was detected in 94% of samples, with a median concentration of 57.63 μg/L. Sb and Li were detected in 72% and 79% of samples at median concentrations of 0.08 μg/L and 0.58 μg/L, respectively. Concentrations of Al, Sb, and Li were not associated with post-partum time. Al was positively associated with total lipid content of samples, weight change since before pregnancy, and coffee and butter intakes and inversely with meat intake. Li was positively associated with intake of chocolate and use of face cream and eyeliner and inversely with year of sample collection, egg, bread, and pasta intakes, and use of hand cream. Sb was positively associated with fatty fish, yoghurt, rice, and deep-fried food intakes and use of eyeliner and inversely with egg and cereal intakes and use of eyeshadow. This study shows that Al, Sb, and Li, especially Al, are widely present in donor breast milk samples. Their concentrations in the milk samples were most frequently associated with dietary habits but also with the lipid content of samples and the use of certain PCPs.

This study developed a sensitive extraction method for urine matrix (based on lyophilization, without the need for pre-cleaning by solid phase extraction), coupled to LC-MS/MS analysis of the biomarker 8-hydroxy-2′ - deoxyguanosine (8-OHdG). The methodology was validated in urine samples from a cohort of Spanish pregnant women collected during the first, second and third trimester of pregnancy, and urine samples collected within 24 h after delivery (n = 85). A detection and quantification limit of 0.01 and 0.05 μg/L, respectively, were established. The median 8-OHdG concentration was 2.18 μg/L (range 0.33–7.79); and the corresponding creatinineadjusted concentrations ranged from 1.04 to 13.12 with median of 4.48 μg 8-OHdG/g creatinine. The concentrations of non-adjusted 8-OHdG significantly decreased (p < 0.05) in the 3rd trimester and post-delivery urine samples when compared to the 1st trimester levels. 8-OHdG concentrations were further studied in placenta samples matching the same urine samples (n = 26), with a median value of 1.3 ng 8-OHdG/g of tissue. Placental 8-OHdG concentrations were correlated with urinary levels of non-adjusted 8-OHdG in the 3rd trimester. Considering the small cohort size, results must be interpreted with caution, however statistical analyses revealed elevated urinary non-adjusted 8-OHdG levels in the 1st trimester of mothers that delivered boys compared to those who delivered girls (p < 0.01). Increased urinary non-adjusted 8-OHdG concentrations at the time of delivery were significantly associated with clinical records (any type of clinical record during pregnancy; p < 0.05). The novel extraction and analytical method for the assessment of 8-OHdG is applicable for sensitive analysis of multiple analytes or biomarkers in urine matrix. This method could also be applied for other matrices such as blood or tissues. Our findings show that 8-OHdG in urine of pregnant women could predict oxidative stress in placenta and can be related to characteristics such as maternal obesity, mode of delivery and newborn sex., European Union through Horizon 2020 projects HBM4EU [grant number 733032], CETOCOEN Excellence [grant number 857560], Horizon Europe project PARC [grant agreement No 101057014], RECETOX Research Infrastructure (No LM2023069), MEYS, and the OP RDE (the CETOCOEN EXCELLENCE project No. CZ.02.1.01/0.0/0.0/ 17_043/0009632)

Introduction: Although often overlooked in clinical settings, accumulation of persistent organic pollutants (POPs) in visceral adipose tissue (VAT) is thought to be a relevant risk factor for metabolic syndrome (MetS). Methods: One hundred and seventeen patients undergoing non-oncological surgery were randomly recruited and classified as MetS + if presented 3 out of the 5 MetS components: waist circumference (WC), systolic and diastolic blood pressure (SBP and DBP, respectively), serum glucose, insulin, triglycerides (TG) and high-density lipoprotein (HDL) cholesterol levels, according International Diabetes Federation (IDF) criteria. Seventeen organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) were measured in adipose tissue samples. Linear, logistic and weighted quantile sum (WQS) regression models, adjusted for age and sex, were performed. Results: One third of the participants were males (36.8%) with a median age of 44 years, showing clinical evidences of MetS (35.0%). Adjusted linear regression models showed that WC correlated positively with all OCP concentrations. Higher fasting serum glucose levels were related to higher HCB and γ-HCH concentrations. The remaining OCPs and PCBs were not associated with this MetS component. HCB was inversely associated with HDL cholesterol levels, while PCB-180 was positively associated. HCB and γ-HCH concentrations were also positively correlated with DBP and SBP levels. PCB-138 was also positively associated with SBP. Adjusted logistic models revealed that exposure to HCB and γ-HCH were associated with increased odds of MetS [ORs (95%CI) 1.53 (1.22–1.92) and 1.39 (1.10–1.76) respectively; p < 0.01]. No associations were observed for the remaining POPs. WQS models showed a positive and significant mixture effect of POPs on the odds of MetS (exp [beta] = 2.34; p < 0.001), with γ-HCH (52.9%), o,p’-DDT (26.9%) and HCB (19.7%) driving the association. Conclusions: Our findings support that POPs accumulated in VAT, specifically HCB and (gamma)-HCH, are associated with both isolated components and clinically diagnosed SMT., HBM4EU under Grant Agreement #733032, Biomedical Research Networking Center-CIBER de Epidemiología y Salud Pública (CIBERESP) of the Institute of Health Carlos III, Institute of Health Carlos III—supported by the European Regional Development Fund/FEDER (FIS-PI16/01820, FIS-PI16/01812 and FIS-PI16/01858), Junta de Andalucía-Consejería de Salud (PS-0506-2016), Ramon y Cajal Program (RYC-2016-20155, Ministerio de Economía, Industria y Competitividad, Spain), (FPU17/01848)

This research would not have been achieved without the selfless col-laboration of the INMA-Granada boys and families who took part in the study. Vicente Mustieles, Alicia Olivas-Martinez and Shereen Cynthia D'Cruz were under contract within the HBM4EU project. Additionally, we acknowledge the Biomedical Research Networking Center-CIBER de Epidemiologia y Salud Publica (CIBERESP) , and the Instituto de Salud Carlos III (ISCIII) (FIS-PI16/01820, FIS-PI16/01858, FIS-PI17/01526, and FIS-PI20/01568) . The authors also thank the ISCIII and "Fondo Europeo de Desarrollo Regional" (ISCIII/FEDER) for the Miguel Servet Type I Program granted to C. Freire (grant no. MS16/00085) , the Sara Borrell postdoctoral research contract granted to F. Vela-Soria (grant no. CD17/00212) , and the Spanish Ministry of Education for the predoctoral fellowships (FPU) granted to A. Rodriguez-Carrillo (FPU 16/03011) and to I. Reina-Perez (FPU 17/01848) . Dr. JP Arrebola is under contract within the Ramon y Cajal Program (RYC-2016-20155, from Ministerio de Economia, Industria y Competitividad, Spain) . The authors also ac-knowledge the contribution of the Pediatric Unit of San Cecilio University Hospital of Granada (recruitment and clinical evaluation) , Marina Molina (field work and biospecimen processing) , Raquel Quesada and Beatriz Suarez (chemical exposure data) and Mario Murcia (data curation) , as well as the Human Genotyping Laboratory at the Spanish National Cancer Research Centre, CeGen-PRB3, which is sup-ported by grant no. PT17/0019, of the PE I + D + i 2013-2016, funded by Instituto de Salud Carlos III (ISCIII) and ERDF. This article will be part of the doctoral thesis developed by Andrea Rodriguez-Carrillo in the context of the "Clinical Medicine and Public Health Program" of the University of Granada (Spain) ., This study was supported in part by the European Union's Horizon 2020 research and innovation program HBM4EU [Grant Agreement No. 733032], Biomedical Research Networking Center-CIBER de Epidemiología y Salud Pública (CIBERESP), and the Instituto de Salud Carlos III (ISCIII) [Grant no. CP16/00085 and FIS-PI17/01526]. The funders had no role in the study design, data collection or analysis, decision to publish, or preparation of the manuscript. Funding for open access charge: Universidad de Granada / CBUA., Background: Bisphenol A (BPA) exposure has been linked to altered behavior in children. Within the European Human Biomonitoring Initiative (HBM4EU), an adverse outcome pathway (AOP) network was constructed supporting the mechanistic link between BPA exposure and brain-derived neurotrophic factor (BDNF). Objective: To test this toxicologically-based hypothesis in the prospective INMA-Granada birth cohort (Spain). Methods: BPA concentrations were quantified by LC-MS/MS in spot urine samples from boys aged 9-11 years, normalized by creatinine and log-2 transformed. At adolescence (15-17 years), blood and urine specimens were collected, and serum and urinary BDNF protein levels were measured using immunoassays. DNA methylation levels at 6 CpGs in Exon IV of the BDNF gene were also assessed in peripheral blood using bisulfite-pyrosequencing. Adolescent's behavior was parent-rated using the Child Behavior Checklist (CBCL/6-18) in 148 boys. Adjusted linear regression and mediation models were fit. Results: Childhood urinary BPA concentrations were longitudinally and positively associated with thought problems (beta = 0.76; 95% CI: 0.02, 1.49) and somatic complaints (beta = 0.80; 95% CI: -0.16, 1.75) at adolescence. BPA concentrations were positively associated with BDNF DNA methylation at CpG6 (beta = 0.21; 95% CI: 0.06, 0.36) and mean CpG methylation (beta = 0.10; 95% CI: 0.01, 0.18), but not with total serum or urinary BDNF protein levels. When independent variables were categorized in tertiles, positive dose-response associations were observed between BPA-thought problems (p-trend = 0.08), BPA-CpG6 (p-trend, Instituto de Salud Carlos III, "Fondo Europeo de Desarrollo Regional" (ISCIII/FEDER) MS16/00085, Sara Borrell postdoctoral research contract grant CD17/00212, Spanish Government FPU 16/03011- FPU 17/01848, Ramon y Cajal Program (Ministerio de Economia, Industria y Competitividad, Spain) RYC-2016-20155, Human Genotyping Laboratory at the Spanish National Cancer Research Centre, CeGen-PRB3 PT17/0019, Instituto de Salud Carlos III European Commission, European Commission

This research would not have been achieved without the selfless collaboration of the INMA-Granada adolescents and families who took part in the study. The authors acknowledge the European Union's Horizon 2020 research and innovation program HBM4EUfor the financial support under Grant Agreement #733032. The study was also supported by the ISCIII with grants no. CP16/00085 and FIS 17/01526, and the Human Genotyping Laboratory at the Spanish National Cancer Research Center (CeGen-PRB3). The authors also thank the support of the Biomedical Research Networking Center-CIBER de Epidemiologia y Salud Publica (CIBERESP), and the University of Granada (Grant "UNETE," UCE-PP2017-06). A. Rodriguez-Carrillo received a predoctoral fellowship (FPU 16/03011) from the Spanish Ministry of Education, Spain, and Vicente Mustieles and Shereen Cynthia D'Cruz were under contract with the HBM4EUproject. C. Freire received a grant under the Miguel Servet Type I program of the ISCIII "Fondo Europeo de Desarrollo Regional" (MS16/00085; ISCIII/FEDER). This article forms part of the doctoral thesis developed by Andrea Rodriguez-Carrillo in the context of the "Clinical Medicine and Public Health Program" of the University of Granada (Spain)., Background: Numerous contemporary non-persistent pesticides may elicit neurodevelopmental impairments. Brain-derived neurotrophic factor (BDNF) has been proposed as a novel effect biomarker of neurological function that could help to understand the biological responses of some environmental exposures. Objectives: To investigate the relationship between exposure to various non-persistent pesticides, BDNF, and behavioral functioning among adolescents. Methods: The concentrations of organophosphate (OP) insecticide metabolites 3,5,6-trichloro-2-pyridinol (TCPy), 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMPy), malathion diacid (MDA), and diethyl thiophosphate (DETP); metabolites of pyrethroids 3-phenoxybenzoic acid (3-PBA) and dimethylcyclopropane carboxylic acid (DCCA), the metabolite of insecticide carbaryl 1-naphthol (1-N), and the metabolite of ethylene-bis-dithiocarbamate fungicides ethylene thiourea (ETU) were measured in spot urine samples, as well as serum BDNF protein levels and blood DNA methylation of Exon IV of BDNF gene in 15–17-year-old boys from the INMA-Granada cohort in Spain. Adolescents’ behavior was reported by parents using the Child Behavior Check List (CBCL/ 6–18). This study included 140 adolescents of whom 118 had data on BDNF gene DNA methylation. Multivariable linear regression, weighted quantile sum (WQS) for mixture effects, and mediation models were fit. Results: IMPy, MDA, DCCA, and ETU were detected in more than 70% of urine samples, DETP in 53%, and TCPy, 3-PBA, and 1-N in less than 50% of samples. Higher levels of IMPy, TCPy, and ETU were significantly associated with more behavioral problems as social, thought problems, and rule-breaking symptoms. IMPy, MDA, DETP, and 1-N were significantly associated with decreased serum BDNF levels, while MDA, 3-PBA, and ETU were associated with higher DNA methylation percentages at several CpGs. WQS models suggest a mixture effect on more behavioral problems and BDNF DNA methylation at several CpGs. A mediated effect of serum BDNF within IMPy-thought and IMPy-rule breaking associations was suggested. Conclusion: BDNF biomarkers measured at different levels of biological complexity provided novel information regarding the potential disruption of behavioral function due to contemporary pesticides, highlighting exposure to diazinon (IMPy) and the combined effect of IMPy, MDA, DCCA, and ETU. However, further research is warranted., European Union's Horizon 2020 research and innovation program HBM4EU 733032, Instituto de Salud Carlos III CP16/00085 FIS 17/01526, Human Genotyping Laboratory at the Spanish National Cancer Research Center (CeGen-PRB3), Biomedical Research Networking Center-CIBER de Epidemiologia y Salud Publica (CIBERESP), University of Granada UCE-PP2017-06, Spanish Government FPU 16/03011, Miguel Servet Type I program of the ISCIII "Fondo Europeo de Desarrollo Regional" (ISCIII/FEDER) MS16/00085

Background. Several non-persistent pesticides are endocrine disrupting chemicals and may impact on sexual maturation. Objective. To examine the association between urinary biomarkers of non-persistent pesticides and sexual maturation in adolescent males in the Environment and Childhood (INMA) Project. Methods. The metabolites of several pesticides were measured in spot urine samples collected from 201 boys aged 14–17 years, including: 3,5,6-trichloro-2-pyridinol (TCPy), metabolite of chlorpyrifos; 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMPy), metabolite of diazinon; malathion diacid (MDA), metabolite of malathion; diethyl thiophosphate (DETP) and diethyl dithiophosphate, non-specific metabolites of organophosphates; 3-phenoxybenzoic acid (3-PBA) and dimethyl cyclopropane carboxylic acid, metabolites of pyrethroids; 1-naphthol (1-NPL), metabolite of carbaryl; and ethylene thiourea (ETU), metabolite of dithiocarbamate fungicides. Sexual maturation was assessed using Tanner stages, self-reported Pubertal Development Scale, and testicular volume (TV). Multivariate logistic regression was employed to examine associations between urinary pesticide metabolites and the odds of being in Tanner stage 5 of genital development (G5) or pubic hair growth (PH5); stage ≥4 of overall pubertal development, gonadarche, and adrenarche; or having mature TV (≥25 mL). Results. DETP concentrations>75th percentile (P75) were associated with lower odds of being in stage G5 (OR = 0.27; 95% CI = 0.10–0.70), detectable TCPy with lower odds of gonadal stage≥4 (OR = 0.50; 95% CI = 0.26–0.96), and intermediate detectable MDA concentrations (, CIBER Epidemiologia y Salud pública, Fondo de investigaciones sanitarias del Instituto Carlos III (código de expediente: PI17/01526), Fondo Europeo de Desarrollo Regional (FEDER) (código de expediente: CP16/00085)

Breast cancer is the most frequently diagnosed cancer and also one of the leading causes of mortality among women. The genetic and environmental factors known to date do not fully explain the risk of developing this disease. In recent years, numerous studies have highlighted the dual role of the gut microbiota in the preservation of host health and in the development of different pathologies, cancer among them. Our gut microbiota is capable of producing metabolites that protect host homeostasis but can also produce molecules with deleterious effects, which, in turn, may trigger inflammation and carcinogenesis, and even affect immunotherapy. The purpose of this review is to describe the mechanisms by which the gut microbiota may cause cancer in general, and breast cancer in particular, and to compile clinical trials that address alterations or changes in the microbiota of women with breast cancer., Junta de Andalucia PI-0538-2017 B-CTS-254-UGR18 B-CTS-254-UGR18 RPS 24665, Programa Operativo FEDER Andalucia 2014-2020, Junta de Andalucia, Spain RPS 24665

This study assessed cross-sectional associations between urinary metabolites of non-persistent pesticides and pubertal development in boys and girls from urban and rural areas in Spain and examined effect modification by body mass index (BMI). Four metabolites of insecticides (TCPy, metabolite of chlorpyrifos; IMPy, metabolite of diazinon; DETP, non-specific metabolite of organophosphates; 3-PBA, metabolite of pyrethroids) and the metabolite of ethylene-bis-dithiocarbamate fungicides (ETU) were quantified in urine collected in 2010-2016 from 7 to 11-year-old children (606 girls, 933 boys) participating in the INMA Project. Pubertal development was ascertained by Tanner stages and/or parent-reported Pubertal Development Scale (PDS). Associations between pesticide metabolites and odds of being in stage 2+ for breast development (girls), genital development (boys), pubic hair growth (girls and boys), and/or overall puberty onset, gonadarche, and adrenarche (PDS for girls and boys) were examined by mixed-effect logistic regression. Effect modification by BMI was explored by interaction terms and stratified analysis. In girls, DETP and ETU concentrations75th percentile (P75) were associated with higher odds of overall puberty development (OR [95%CI] = 1.86 [1.07-3.24] and 1.71 [1.03-2.83], respectively, for P75 vs. undetected concentrations), while ETU P75 was also associated with higher odds of breast development (OR [95%CI] = 5.55 [2.83-12.91]), particularly in girls with underweight/normal weight (OR [95%CI] = 10.08 [2.62-38.76]). In boys, detection of TCPy (40%) and 3-PBA (34%) was associated with higher odds of genital development (OR [95%CI] = 1.97 [1.08-3.57] and 2.08 [1.15-3.81], respectively), and the association with 3-PBA was observed in boys with overweight/obesity alone. In addition, ETU P75 was associated with higher odds of genital development in boys with underweight/normal weight (OR [95%CI] = 2.89 [1.08-7.74]) but higher DETP with lower odds of puberty in boys with overweight/obesity (OR [95%CI] = 0.94 [0.89-0.99] per log-unit increase in concentration). Results suggest an association of childhood exposure to ETU and certain insecticides with earlier puberty in girls and boys that may be modified by child BMI.

This research would not have been achieved without the selfless collaboration of the INMA-Granada adolescents and families who took part in the study. The authors also acknowledge the Human Genotyping Laboratory at the Spanish National Cancer Research Center, CeGen-PRB3, which is supported by grant no. PT17/0019, of the PE I+D+i 2013-2016, funded by the Instituto de Salud Carlos III (ISCIII) and ERDF. They thank the European Union's Horizon 2020 research and innovation program HBM4EU for financial support under Grant Agree-ment No. 733032. The study was also supported by the ISCIII with grant no. CP16/00085. The authors also acknowledge the funding received from the University of Granada for the open access publishing costs and the support of the Biomedical Research Networking Center-CIBER de Epidemiologia y Salud Publica (CIBERESP) , and the ISCIII (FIS 17/01526, FIS-PI16/01820 and FIS-PI16/01858) . Vicente Mustieles and Shereen Cynthia D'Cruz were under contract with the HBM4EU project. A. Rodriguez-Carrillo received a predoctoral fellowship (FPU 16/03011) from the Spanish Ministry of Education and C. Freire (grant no. MS16/00085) received a grant under the Miguel Servet Type I program of the ISCIII "Fondo Europeo de Desarrollo Regional" (ISCIII/FEDER) . This article forms part of the doctoral thesis developed by Andrea Rodriguez-Carrillo in the context of the "Clinical Medicine and Public Health Program" of the University of Granada (Spain) ., Background: Brain-derived neurotrophic factor (BDNF) plays an important role in brain development by regulating multiple pathways within the central nervous system. In the Human Biomonitoring for Europe Project (HBM4EU), this neurotrophin is being implemented as a novel effect biomarker to evaluate the potential threats of environmental chemicals on neurodevelopment. Objectives: To explore the relationships among exposure to environmental metals, BDNF biomarkers at two levels of biological complexity, and behavioral function in adolescent males. Methods: Data were gathered from 125 adolescents on: spot urine sample total concentrations of the neurotoxic metal(oid)s arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb); serum BDNF protein concentrations; and concurrent behavioral functioning according to the Child Behavior Check List (CBCL/6–18). In 113 of the participants, information was also collected on blood BDNF DNA methylation at six CpGs. Associations were evaluated by multivariate linear regression analysis adjusted for confounders. Results: As, Cd, Hg, and Pb were detected in 100%, 98.5%, 97.0%, and 89.5% of urine samples, respectively. Median serum BDNF concentration was 32.6 ng/mL, and total percentage of BDNF gene methylation was 3.8%. In the adjusted models, urinary As was non-linearly associated with more internalizing problems and Cd with more externalizing behaviors. The percentage BDNF DNA methylation at CPGs #5 and the mean percentage CpG methylation increased across As tertiles (p-trend = 0.04 and 0.03, respectively), while 2nd tertile and 3rd tertile of Cd concentrations were associated with lower serum BDNF and higher CpG3 methylation percentage. Additionally, when BDNF was categorized in tertiles, serum BDNF at the 3rd tertile was associated with fewer behavioral problems, particularly withdrawn (p-trend = 0.04), social problems (p-trend = 0.12), and thought problems (p-trend = 0.04). Conclusion: Exposure to As and Cd was associated with BDNF gene DNA methylation BDNF gene and serum BDNF, respectively. Associations with DNA methylation may be attributable to a higher variability over time in circulating BDNF concentrations than in the methylation status of this gene. Caution should be taken when interpreting the results relating postnatal Pb and Hg to behavioral functioning. Further studies are needed to verify these findings., Instituto de Salud Carlos III European Commission PT17/0019, European Commission, European Union's Horizon 2020 research and innovation program HBM4EU 733032, Instituto de Salud Carlos III CP16/00085 FIS 17/01526 FIS-PI16/01820 FIS-PI16/01858, University of Granada, Biomedical Research Networking Center-CIBER de Epidemiologia y Salud Publica (CIBERESP), Spanish Government FPU 16/03011, Miguel Servet Type I program of the ISCIII "Fondo Europeo de Desarrollo Regional" (ISCIII/FEDER) MS16/00085

Higher maternal pre-pregnancy body mass index (ppBMI) is associated with increased neonatal morbidity, as well as with pregnancy complications and metabolic outcomes in offspring later in life. The placenta is a key organ in fetal development and has been proposed to act as a mediator between the mother and different health outcomes in children. The overall aim of the present work is to investigate the association of ppBMI with epigenomewide placental DNA methylation (DNAm) in 10 studies from the PACE consortium, amounting to 2631 mother-child pairs. We identify 27 CpG sites at which we observe placental DNAm variations of up to 2.0% per 10 ppBMI-unit. The CpGs that are differentially methylated in placenta do not overlap with CpGs identified in previous studies in cord blood DNAm related to ppBMI. Many of the identified CpGs are located in open sea regions, are often close to obesity-related genes such as GPX1 and LGR4 and altogether, are enriched in cancer and oxidative stress pathways. Our findings suggest that placental DNAm could be one of the mechanisms by which maternal obesity is associated with metabolic health outcomes in newborns and children, although further studies will be needed in order to corroborate these findings., French Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences de l'Univers (INSU), Swiss National Science Foundation (SNSF), European Commission, Ministry of Science and Innovation, Spain (MICINN), Spanish Government FJC2018-036729, European Development Fund, European Social Fund (ESF)

Background Bisphenol A (BPA) has been associated with impairments in children’s behavior, but few studies have investigated its relationship with cognitive function. Objective To investigate the association of urinary BPA concentrations with cognitive domains and intelligence quotient (IQ) in Spanish boys. Methods BPA levels were quantified by liquid chromatography-tandem mass spectrometry (LC–MS-MS) in one spot urine sample from 269 boys of the INMA-Granada cohort, in their follow-up at 9–11 years of age. Cognitive function was evaluated by a trained psychologist using a comprehensive neuropsychological test battery (including general intelligence, language skills, working memory, attention, impulsivity, visual-motor coordination, processing speed and executive function, among others). Cross-sectional associations between BPA levels and neuropsychological standardized scores were analyzed by adjusted linear and logistic regression models. Results Median (P25, P75) BPA concentrations were 4.76 (2.77, 9.03) μg/L and 4.75 (2.75, 10.2) μg/g of creatinine (Cr). Boys in the third and fourth quartile of volume-based BPA concentrations showed better processing speed scores than boys in the first quartile (β = 5.47; 95%CI: 1.4, 9.4 and β = 3.57; 95%CI: -0.4, 7.5, respectively); and boys in the third quartile showed better inhibitory control (β = 1.6; 95%CI: -0.3, 3.5) and impulsivity (β= -4.2; 95%CI: -9.0, 0.0). In contrast, boys in the fourth quartile showed poorer working memory scores than those in the first quartile (β= -1.0; 95%CI: -2.1, -0.1). All these associations were attenuated when Cr-standardized BPA concentrations were considered. Cr-based BPA concentrations were also associated with a higher risk of being below the 20th percentile of working memory scores [ORa = 1.51; 95%CI: 1.01, 2.25]. Discussion Our findings do not support an association between urinary BPA concentrations and cognitive function or IQ among boys, except for working memory. BPA was previously found to be associated with behavior problems in the same study population, suggesting that BPA may predominantly affect the behavior of children rather than their cognitive function, in line with previous epidemiologic studies.

To examine the association of placental levels of arsenic (As), cadmium (Cd), mercury (Hg), lead (Pb), manganese (Mn), and chromium (Cr) with birth outcomes (birth weight, length, and head circumference, low birth weight [LBW], gestational age, preterm delivery, and small for gestational age [SGA]) in mother-child pairs from the Environment and Childhood (INMA) Project in Spain.Metal concentrations were measured in placenta tissue samples randomly selected from five INMA cohorts. Data on birth outcomes were obtained from medical records. Associations were assessed in a sub-sample of 327 mother-infant pairs by regression models adjusted for confounding factors and for all metals simultaneously. Effect modification by sex was also evaluated.Elevated placental Cd levels (5.79 vs.3.30 ng/g) were associated with reduced birth weight (-111.8 g, 95%CI = -215.6; -8.06, p-trend = 0.01) and length (-0.62 cm, 95%CI = -1.20; -0.04, p-trend = 0.02), while a 10% increase in Cd was associated with 1.21-fold increased odds (95%CI = 1.01; 1.43) of LBW in the global sample but with 14% lower odds (95%CI = 0.78; 0.96) of preterm delivery in males (PData suggest that in utero exposure to Cd, Hg, and Cr could adversely affect fetal growth, whereas Mn and Cr appear to have a positive effect on gestational age. Given the relatively small number of subjects, sex-specific associations should be interpreted with caution.

© 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)., Data generated by the rapidly evolving human biomonitoring (HBM) programmes are providing invaluable opportunities to support and advance regulatory risk assessment and management of chemicals in occupational and environmental health domains. However, heterogeneity across studies, in terms of design, terminology, biomarker nomenclature, and data formats, limits our capacity to compare and integrate data sets retrospectively (reuse). Registration of HBM studies is common for clinical trials; however, the study designs and resulting data collections cannot be traced easily. We argue that an HBM Global Registry Framework (HBM GRF) could be the solution to several of challenges hampering the (re)use of HBM (meta)data. The aim is to develop a global, host-independent HBM registry framework based on the use of harmonised open-access protocol templates from designing, undertaking of an HBM study to the use and possible reuse of the resulting HBM (meta)data. This framework should apply FAIR (Findable, Accessible, Interoperable and Reusable) principles as a core data management strategy to enable the (re)use of HBM (meta)data to its full potential through the data value chain. Moreover, we believe that implementation of FAIR principles is a fundamental enabler for digital transformation within environmental health. The HBM GRF would encompass internationally harmonised and agreed open access templates for HBM study protocols, structured web-based functionalities to deposit, find, and access harmonised protocols of HBM studies. Registration of HBM studies using the HBM GRF is anticipated to increase FAIRness of the resulting (meta)data. It is also considered that harmonisation of existing data sets could be performed retrospectively. As a consequence, data wrangling activities to make data ready for analysis will be minimised. In addition, this framework would enable the HBM (inter)national community to trace new HBM studies already in the planning phase and their results once finalised. The HBM GRF could also serve as a platform enhancing communication between scientists, risk assessors, and risk managers/policy makers. The planned European Partnership for the Assessment of Risk from Chemicals (PARC) work along these lines, based on the experience obtained in previous joint European initiatives. Therefore, PARC could very well bring a first demonstration of first essential functionalities within the development of the HBM GRF.

The 3T3-L1 murine pre-adipocyte line is an established cell culture model for screening Metabolism Disrupting Chemicals (MDCs). Despite a need to accurately identify MDCs for further evaluation, relatively little research has been performed to comprehensively evaluate reproducibility across laboratories, assess factors that might contribute to varying degrees of differentiation between laboratories (media additives, plastics, cell source, etc.), or to standardize protocols. As such, the goals of this study were to assess interlaboratory variability of efficacy and potency outcomes for triglyceride accumulation and pre-adipocyte proliferation using the mouse 3T3-L1 pre-adipocyte cell assay to test chemicals. Ten laboratories from five different countries participated. Each laboratory evaluated one reference chemical (rosiglitazone) and three blinded test chemicals (tributyltin chloride, pyraclostrobin, and bisphenol A) using: 1) their Laboratory-specific 3T3-L1 Cells (LC) and their Laboratory-specific differentiation Protocol (LP), 2) Shared 3T3-L1 Cells (SC) with LP, 3) LC with a Shared differentiation Protocol (SP), and 4) SC with SP. Blinded test chemical responses were analyzed by the coordinating laboratory. The magnitude and range of bioactivities reported varied considerably across laboratories and test conditions, though the presence or absence of activity for each tested chemical was more consistent. Triglyceride accumulation activity determinations for rosiglitazone ranged from 90 to 100% across test conditions, but 30–70 % for pre-adipocyte proliferation; this was 40–80 % for triglyceride accumulation induced by pyraclostrobin, 80–100 % for tributyltin, and 80–100 % for bisphenol A. Consistency was much lower for pre-adipocyte proliferation, with 30–70 % active determinations for pyraclostrobin, 30–50 % for tributyltin, and 20–40 % for bisphenol A. Greater consistency was observed for the SC/SP assessment. As such, working to develop a standardized adipogenic differentiation protocol represents the best strategy for improving consistency of adipogenic responses using the 3T3-L1 model to reproducibly identify MDCs and increase confidence in reported outcomes. Over-arching project supported by grants [R01 ES016099 to HMS; R00 ES030405 to CDK] from the National Institute of Environmental Health Sciences (NIEHS); University of Turin; European Union's Horizon 2020 research and innovation program under grant agreement GOLIATH No. 825489; Brunel University London; NIEHS (1K22ES026208 and R01ES027863); NIEHS (Z0ES102785); Spanish Institute of Health Carlos III (grant FIS-PI16/01812).

We would like to thank all the families that participated in these studies for their generous contribution. Detailed acknowledgements and funding can be found in Sup plementary Material., Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-021-24558-y, Maternal smoking during pregnancy contributes to poor birth outcomes. Here the authors perform a meta-analysis of the associations between maternal smoking during pregnancy and placental DNA methylation and identify links between these and poor birth outcomes, which may better inform the mechanisms through which smoking impacts placental function and fetal growth. Maternal smoking during pregnancy (MSDP) contributes to poor birth outcomes, in part through disrupted placental functions, which may be reflected in the placental epigenome. Here we present a meta-analysis of the associations between MSDP and placental DNA methylation (DNAm) and between DNAm and birth outcomes within the Pregnancy And Childhood Epigenetics (PACE) consortium (N = 1700, 344 with MSDP). We identify 443 CpGs that are associated with MSDP, of which 142 associated with birth outcomes, 40 associated with gene expression, and 13 CpGs are associated with all three. Only two CpGs have consistent associations from a prior meta-analysis of cord blood DNAm, demonstrating substantial tissue-specific responses to MSDP. The placental MSDP-associated CpGs are enriched for environmental response genes, growth-factor signaling, and inflammation, which play important roles in placental function. We demonstrate links between placental DNAm, MSDP and poor birth outcomes, which may better inform the mechanisms through which MSDP impacts placental function and fetal growth., Canadian Institutes of Health Research (CIHR) MOP 115071, United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of Environmental Health Sciences (NIEHS) P30 ES019776 - R01 ES022223 - P01 ES022832, United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of General Medical Sciences (NIGMS) P20 GM104416, United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of Mental Health (NIMH) R01 MH094609

This study has been partially funded by PID2019-107036RB-I00, from Ministerio de Ciencia, Innovacion y Universidades, Madrid, Spain, and 2020-0287, from IIS ISABIAL, Hospital General Universitario, Alicante, Spain. RL is recipient of a grant (FPU 18/00063) by Ministerio de Ciencia, Innovacion y Universidades, Madrid, Spain., The relevance of environmental triggers in Crohn's disease remains poorly explored, despite the well-known association between industrialization and disease onset/progression. We have aimed at evaluating the influence of endocrine disrupting chemicals in CD patients. We performed a prospective observational study on consecutive patients diagnosed of CD. Serum levels of endocrine disruptors, short-chain fatty acids, tryptophan and cytokines were measured. Bacterial-DNA and serum endotoxin levels were also evaluated. Gene expression of ER-α, ER-β and GPER was measured in PBMCs. All patients were genotyped for NOD2 and ATG16L1 polymorphisms. A series of 200 CD patients (140 in remission, 60 with active disease) was included in the study. Bisphenol A was significantly higher in patients with active disease versus remission and in colonic versus ileal disease. GPER was significantly increased in active patients and correlated with BPA levels. BPA was significantly increased in patients with bacterial-DNA and correlated with serum endotoxin levels, (r = 0.417; P = .003). Serum butyrate and tryptophan levels were significantly lower in patients with bacterial-DNA and an inverse relationship was present between them and BPA levels (r = −0.491; P = .001) (r = −0.611; P = .001). Serum BPA levels correlated with IL-23 (r = 0.807; P = .001) and IL-17A (r = 0.743; P = .001). The multivariate analysis revealed an independent significant contribution of BPA and bacterial-DNA to serum levels of IL-23 and IL-17A. In conclusion, bisphenol A significantly affects systemic inflammatory response in CD patients with gut barrier disruption and dysbiotic microbiota secretory products in blood. These results provide evidence of an endocrine disruptor playing an actual pathogenic role on CD., Ministerio de Ciencia, Innovacion y Universidades, Madrid, Spain PID2019-107036RB-I00 FPU 18/00063, IIS ISABIAL, Hospital General Universitario, Alicante, Spain 2020-0287

Objective: To assess the relationship of urinary concentrations of ethylenethiourea (ETU), the main degradation product of ethylene bis-dithiocarbamate fungicides, 3-phenoxybenzoic acid (3-PBA), a common metabolite of many pyrethroids, and 1-naphthol (1N), a metabolite of the carbamate insecticide carbaryl, with hormone concentrations in adolescent males; and to examine interactions between pesticide metabolites and polymorphisms in xenobiotic metabolizing enzymes, including CYP2C19 and CYP2D6, in relation to hormone concentrations. Methods: A cross-sectional study was conducted in 134 males from the Spanish Environment and Childhood (INMA)-Granada cohort. Urine and serum samples were collected from participants during the same clinical visit at the age of 15–17 years. First morning urine void was analyzed for concentrations of ETU, 3-PBA, and 1N. Serum was analyzed for concentrations of reproductive hormones (testosterone, 17β-estradiol [E2], dehydroepiandrosterone sulfate [DHEAS], sex hormone binding globulin [SHBG], luteinizing hormone [LH], follicle stimulating hormone [FSH], anti-Müllerian hormone [AMH], and prolactin), thyroid hormones (free thyroxine [FT4], total triiodothyronine [TT3], and thyroid stimulating hormone [TSH]), insulin growth factor 1 (IGF-1), adrenocorticotropic hormone (ACTH), and cortisol. CYP2C19 G681A and CYP2D6 G1846A polymorphisms were determined in blood from 117 participants. Multiple linear regression, interaction terms, and stratified analyses were performed. Results: Urinary ETU was detected in 74.6% of participants, 1N in 38.1%, and 3-PBA in 19.4%. Positive associations between detectable 3-PBA and TT3 and between detectable 1N and DHEAS were found, and marginally-significant associations of 1N with reduced E2 and FSH were observed. Poor CYP2C19 and CYP2D6 metabolizers (GA and AA genotype carriers) showed a greater increase in DHEAS for detected versus undetected 1N compared with GG genotype carriers. Poor CYP2D6 metabolizers (1846 GA and AA genotypes) evidenced increased cortisol for detected versus undetected ETU. Conclusions: The associations observed between urinary pesticide metabolites and altered thyroid and reproductive hormones are novel and should be verified in studies with larger sample size. Further research on gene-environment interactions is warranted to establish individual susceptibility to pesticides and the risk of adverse health effects.

This work was supported by research grants from CIBER de Epidemiologia y Salud Publica (CIBERESP), Instituto de Salud Carlos III, Junta de Andalucia, European Regional Development Fund-FEDER (PI16/01858, PI16/01812, PI20/01568 and PI-13/02406) and Generalitat Valenciana. Dr. G Cakmak is awarded a grant by The Scientific and Technological Research Council of Turkey (TUBITAK-2219). Dr. JP Arrebola is under contract within the Ramón y Cajal Program (RYC-2016-20155, Ministerio de Economia, Industria y Competitividad, Spain)., These results would not have been achieved without the selfless collaboration of the staff from Santa Ana and San Cecilio Hospitals and the participants who took part in the study., Humans are exposed to a myriad of chemical substances in both occupational and environmental settings. Persistent organic pollutants (POPs) have drawn attention for their adverse effects including cancer and endocrine disruption. Herein, the objectives were 1) to describe serum and adipose tissue retinol levels, along with serum retinol binding protein 4 (RBP4) concentrations, and 2) to assess the associations of adipose tissue POP levels with these retinoid parameters, as well as their potential interaction with the previously-observed POP-related disruption of redox microenvironment. Retinol was measured in both serum and adipose tissue along with RBP4 levels in serum samples of 236 participants of the GraMo adult cohort. Associations were explored by multivariable linear regression analyses and Weighted Quantile Sum regression. Polychlorinated biphenyls (PCBs) 180, 153 and 138 were related to decreased adipose tissue retinol levels and increased serum RBP4/retinol ratio. Dicofol concentrations > limit of detection were associated with decreased retinol levels in serum and adipose tissue. Additionally, increased adipose tissue retinol levels were linked to an attenuation in previously-reported associations of adipose tissue PCB-153 with in situ superoxide dismutase activity. Our results revealed a suggestive link between retinoids, PCBs and redox microenvironment, potentially relevant for both mechanistic and public health purposes., CIBER de Epidemiologia y Salud Publica (CIBERESP), Instituto de Salud Carlos III, Junta de Andalucia, European Commission PI16/01858 PI16/01812 PI20/01568 PI-13/02406, Generalitat Valenciana, European Commission, Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) TUBITAK-2219, Ramon y Cajal Program (Ministerio de Economia, Industria y Competitividad, Spain) RYC-2016-20155