Association of exposure to perfluoroalkyl substances (PFAS) and phthalates with thyroid hormones in adolescents from HBM4EU aligned studies.

BACKGROUND: Perfluoroalkyl substances (PFAS) and phthalates are synthetic chemicals widely used in various types of consumer products. There is epidemiological and experimental evidence that PFAS and phthalates may alter thyroid hormone levels; however, studies in children and adolescents are limited. AIM: To investigate the association of exposure to PFAS and phthalate with serum levels of thyroid hormones in European adolescents. METHODS: A cross-sectional study was conducted in 406 female and 327 male adolescents (14-17 years) from Belgium, Slovakia, and Spain participating in the Aligned Studies of the HBM4EU Project (FLEHS IV, PCB cohort, and BEA, respectively). Concentrations of perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorononanoic acid (PFNA), free thyroxine (FT4), free triiodothyronine (FT3), and thyroid-stimulating hormone (TSH) were measured in sera from study participants, and urinary metabolites of six phthalates (DEP, DiBP, DnBP, BBzP, DEHP, and DiNP) and the non-phthalate plasticizer DINCH® were quantified in spot urine samples. Associations were assessed with linear regression and g-computational models for mixtures. Effect modification by sex was examined. RESULTS: In females, serum PFOA and the PFAS mixture concentrations were associated with lower FT4 and higher FT3 levels; MEP and the sums of DEHP, DiNP, and DINCH® metabolites (∑DEHP, ∑DiNP, and ∑DINCH) were associated with higher FT4; ∑DEHP with lower FT3; and the phthalate/DINCH® metabolite mixture with higher FT4 and lower FT3. In males, PFOA was associated with lower FT4 and the PFAS mixture with higher TSH levels and lower FT4/TSH ratio; MEP and ∑DiNP were associated with higher FT4; and MBzP, ∑DEHP, and the phthalate/DINCH® metabolite mixture with lower TSH and higher FT4/TSH. PFOA, mono-(2-ethyl-5-hydroxyhexyl) phthalate (OH-MEHP), mono-(2-ethyl-5-oxohexyl) phthalate (oxo-MEHP), and monocarboxyoctyl phthalate (MCOP) made the greatest contribution to the mixture effect. CONCLUSIONS: Results suggest that exposure to PFAS and phthalates is associated with sex-specific differences in thyroid hormone levels in adolescents.

Biomarkers of metabolic disorders and neurobehavioral diseases in a PCB- exposed population: What we learned and the implications for future research.

Polychlorinated biphenyls (PCBs) are one of the original twelve classes of toxic chemicals covered by the Stockholm Convention on Persistent Organic Pollutants (POP), an international environmental treaty signed in 2001. PCBs are present in the environment as mixtures of multiple isomers at different degree of chlorination. These compounds are manmade and possess useful industrial properties including extreme longevity under harsh conditions, heat absorbance, and the ability to form an oily liquid at room temperature that is useful for electrical utilities and in other industrial applications. They have been widely used for a wide range of industrial purposes over the decades. Despite a ban in production in 1979 in the US and many other countries, they remain persistent and ubiquitous in environment as contaminants due to their improper disposal. Humans, independent of where they live, are therefore exposed to PCBs, which are routinely found in random surveys of human and animal tissues. The prolonged exposures to PCBs have been associated with the development of different diseases and disorders, and they are classified as endocrine disruptors. Due to its ability to interact with thyroid hormone, metabolism and function, they are thought to be implicated in the global rise of obesity diabetes, and their potential toxicity for neurodevelopment and disorders, an example of gene by environmental interaction (GxE). The current review is primarily intended to summarize the evidence for the association of PCB exposures with increased risks for metabolic dysfunctions and neurobehavioral disorders. In particular, we present evidence of gene expression alterations in PCB-exposed populations to construct the underlying pathways that may lead to those diseases and disorders in course of life. We conclude the review with future perspectives on biomarker-based research to identify susceptible individuals and populations.

Environmental ototoxicants, a potential new class of chemical stressors.

Hearing loss is an injury that can develop over time, and people may not even be aware of it until it becomes a severe disability. Ototoxicants are substances that may damage the inner ear by either affecting the structures in the ear itself or by affecting the nervous system. We have examined the possibility that ototoxicants may present a health hazard in association with environmental exposures, adding to existing knowledge of their proven hazards under medical therapeutic conditions or occupational activities. In addition to the already described human environmental ototoxicants, mainly organochlorines such as polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene (DDE), hexachlorocyclohexane (HCH) and hexachlorobenzene (HCB), we have examined the ubiquitous chemical stressors phthalates, bisphenol A/S/F/, PFCs, flame retardants (FRs) and cadmium for potential ototoxic properties, both as single substances or as chemical mixtures. Our literature review confirmed that these chemicals may disturb thyroid hormones homeostasis, activate aryl hydrocarbon receptor (AhR), and induce oxidative stress, which in turn may initiate a chain of events resulting in impairment of cochlea and hearing loss. With regard to auditory plasticity, diagnostics of a mixture of effects of ototoxicants, potential interactions of chemical and physical agents with effects on hearing, parallel deterioration of hearing due to chemical exposures and ageing, metabolic diseases or obesity, even using specific methods as brainstem auditory evoked potentials (BAEP) or otoacoustic emissions (OAEs) registration, may be difficult, and establishment of concentration-response relationships problematic. This paper suggests the establishment of a class of environmental oxotoxicants next to the established classes of occupational and drug ototoxicants. This will help to properly manage risks associated with human exposure to chemical stressors with ototoxic properties and adequate regulatory measures.

Gestational weight gain charts for different body mass index groups for women in Europe, North America, and Oceania.

BACKGROUND: Gestational weight gain differs according to pre-pregnancy body mass index and is related to the risks of adverse maternal and child health outcomes. Gestational weight gain charts for women in different pre-pregnancy body mass index groups enable identification of women and offspring at risk for adverse health outcomes. We aimed to construct gestational weight gain reference charts for underweight, normal weight, overweight, and grades 1, 2 and 3 obese women and to compare these charts with those obtained in women with uncomplicated term pregnancies. METHODS: We used individual participant data from 218,216 pregnant women participating in 33 cohorts from Europe, North America, and Oceania. Of these women, 9065 (4.2%), 148,697 (68.1%), 42,678 (19.6%), 13,084 (6.0%), 3597 (1.6%), and 1095 (0.5%) were underweight, normal weight, overweight, and grades 1, 2, and 3 obese women, respectively. A total of 138, 517 women from 26 cohorts had pregnancies with no hypertensive or diabetic disorders and with term deliveries of appropriate for gestational age at birth infants. Gestational weight gain charts for underweight, normal weight, overweight, and grade 1, 2, and 3 obese women were derived by the Box-Cox t method using the generalized additive model for location, scale, and shape. RESULTS: We observed that gestational weight gain strongly differed per maternal pre-pregnancy body mass index group. The median (interquartile range) gestational weight gain at 40 weeks was 14.2 kg (11.4-17.4) for underweight women, 14.5 kg (11.5-17.7) for normal weight women, 13.9 kg (10.1-17.9) for overweight women, and 11.2 kg (7.0-15.7), 8.7 kg (4.3-13.4) and 6.3 kg (1.9-11.1) for grades 1, 2, and 3 obese women, respectively. The rate of weight gain was lower in the first half than in the second half of pregnancy. No differences in the patterns of weight gain were observed between cohorts or countries. Similar weight gain patterns were observed in mothers without pregnancy complications. CONCLUSIONS: Gestational weight gain patterns are strongly related to pre-pregnancy body mass index. The derived charts can be used to assess gestational weight gain in etiological research and as a monitoring tool for weight gain during pregnancy in clinical practice.

Dynamics of allergy development during the first 5 years of life.

Allergic diseases have increased in developed countries during the past decades. A cohort of Slovak children was followed from birth to track allergic symptoms dynamics in early childhood. Information on allergic symptoms (atopic dermatitis = AD, rhino conjunctivitis = RC, wheezing = Wh, urticaria = Ur) and food allergies among children was based on clinical evaluation of children by allergists at three developmental stages (infant, toddler, preschool). Out of 320 cases of allergies, 64 infants, 145 toddlers, and 195 preschool children suffered from AD, RC, Wh, Ur, or their combinations (i.e., significant increase with age, p < 0.001). AD first appeared in infants, Wh and/or RC rose mainly in toddlers, and Ur among preschool children. AD in infants or toddlers disappeared in the subsequent developmental stage in approximately one third of all cases. Single AD persistence without remission or extension was not common and accounted only for 6.9% of AD infants' allergic manifestations. In addition to single-symptom allergic diseases, this study also identified several combinations of atopic symptoms.Conclusions: The proportion of multi-symptom allergies increased while single-symptom forms decreased. The observed temporal trends of allergic symptoms correspond to the atopic march. What is Known: • The observed temporal trends of allergic symptoms correspond to the atopic march. What is New: • Allergic diseases in children were first manifested as single forms, with atopic dermatitis (AD) commonly functioning as the "entry point" to allergies. • The overall proportion of single-symptom allergic disorders decreased over time while the proportion of multi-symptom allergies increased.

Demographic, Reproductive, and Dietary Determinants of Perfluorooctane Sulfonic (PFOS) and Perfluorooctanoic Acid (PFOA) Concentrations in Human Colostrum.

To determine demographic, reproductive, and maternal dietary factors that predict perfluoroalkyl substance (PFAS) concentrations in breast milk, we measured perfluorooctane sulfonic (PFOS) and perfluorooctanoic acid (PFOA) concentrations, using liquid chromatography-mass spectrometry, in 184 colostrum samples collected from women participating in a cohort study in Eastern Slovakia between 2002 and 2004. During their hospital delivery stay, mothers completed a food frequency questionnaire, and demographic and reproductive data were also collected. PFOS and PFOA predictors were identified by optimizing multiple linear regression models using Akaike's information criterion (AIC). The geometric mean concentration in colostrum was 35.3 pg/mL for PFOS and 32.8 pg/mL for PFOA. In multivariable models, parous women had 40% lower PFOS (95% CI: -56 to -17%) and 40% lower PFOA (95% CI: -54 to -23%) concentrations compared with nulliparous women. Moreover, fresh/frozen fish consumption, longer birth intervals, and Slovak ethnicity were associated with higher PFOS and PFOA concentrations in colostrum. These results will help guide the design of future epidemiologic studies examining milk PFAS concentrations in relation to health end points in children.

PCB exposure and cochlear function at age 6 years.

Epidemiological studies have documented adverse associations between exposure to polychlorinated biphenyls (PCBs) and otological outcomes. Previously, we documented decreased distortion product otoacoustic emission (DPOAE) levels in children exposed to PCBs, up to the age of 45 months, amongst a cohort of children in eastern Slovakia. The objective of the present study is to evaluate cochlear dysfunction at 72 months of age in 214 children from this same cohort and to compare the otoacoustic test sensitivity to that of pure tone audiometry (PTA). The association between DPOAE, PTA, and PCBs was estimated by means of multivariate ANOVA (MANOVA) and linear regression models. ROC curves were computed to estimate the DPOAE-test power in children. The DPOAE level at 72 months was related to PCB-153 serum levels. The DPOAE Input/Output function test at mid-frequency (2kHz) has shown instead nonmonotonic dependence on PCB exposure, for the left ears of children, over the whole growth curve. No significant association was found between PTA hearing levels and PCB-153 concentration. High diagnostic power of the DPOAE-test was found in children, similar to that found by the same authors in adults. In conclusions the DPOAE-PCB correlation obtained at 72 months is similar to that at 45 months suggesting a permanent and stable ototoxic effect of the PCB exposure. The lack of statistical significance of the PCB-PTA correlation suggests that DPOAEs are sensitive biomarkers of cochlear damage.

Duration of breastfeeding and serum PCB 153 concentrations in children.

Polychlorinated biphenyls (PCBs) are toxic, persistent, and bioaccumulative chemicals which, because of their lipophilic properties, are abundant in human breast milk. Breastfed infants are therefore at risk of being exposed to considerable amounts of PCBs. The commonly used exposure estimations, based solely on breast milk PCB levels and duration of breastfeeding, may lead to exposure misclassification. To improve assessments of exposure to PCBs, we determined PCB 153 serum concentration, as a model substance for PCBs, at the critical time of weaning for each child in 305 breastfed infants from 5 single time point concentration measurements spread over 7 years and data on duration of breastfeeding, using an earlier developed model of the system type. We approximated the dependence of PCB 153 serum concentration, Ctbf, adjusted to cord serum concentration, C0, on nursing period, by a polynomial function Ctbf/C0=0.596+0.278t-0.0047t(2) which reliably predicts exposure to PCB 153 of breastfed infants, important for assessment of dose-outcome relationships. Adjustment of current serum concentrations to cord serum concentration improved validity of exposure assessment.

Transcriptional profiling and biological pathway analysis of human equivalence PCB exposure in vitro: indicator of disease and disorder development in humans.

BACKGROUND AND AIMS: Our earlier gene-expression studies with a Slovak PCBs-exposed population have revealed possible disease and disorder development in accordance with epidemiological studies. The present investigation aimed to develop an in vitro model system that can provide an indication of disrupted biological pathways associated with developing future diseases, well in advance of the clinical manifestations that may take years to appear in the actual human exposure scenario. METHODS: We used human Primary Blood Mononuclear Cells (PBMC) and exposed them to a mixture of human equivalence levels of PCBs (PCB-118, -138, -153, -170, -180) as found in the PCBs-exposed Slovak population. The microarray studies of global gene expression were conducted on the Affymetrix platform using Human Genome U133 Plus 2.0 Array along with Ingenuity Pathway Analysis (IPA) to associate the affected genes with their mechanistic pathways. High-throughput qRT-PCR Taqman Low Density Array (TLDA) was done to further validate the selected 6 differentially expressed genes of our interest, viz., ARNT, CYP2D6, LEPR, LRP12, RRAD, TP53, with a small population validation sample (n=71). RESULTS: Overall, we revealed a discreet gene expression profile in the experimental model that resembled the diseases and disorders observed in PCBs-exposed population studies. The disease pathways included endocrine system disorders, genetic disorders, metabolic diseases, developmental disorders, and cancers, strongly consistent with the evidence from epidemiological studies. INTERPRETATION: These gene finger prints could lead to the identification of populations and subgroups at high risk for disease, and can pose as early disease biomarkers well ahead of time, before the actual disease becomes visible.

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.

Urinary concentrations of phthalate/DINCH metabolites and body mass index among European children and adolescents in the HBM4EU Aligned Studies: A cross-sectional multi-country study.

BACKGROUND: Phthalates are ubiquitous in the environment. Despite short half-lives, chronic exposure can lead to endocrine disruption. The safety of phthalate substitute DINCH is unclear. OBJECTIVE: To evaluate associations between urinary concentrations of phthalate/DINCH metabolites and body mass index (BMI) z-score among children and adolescents. METHOD: We used Human Biomonitoring for Europe Aligned Studies data from 2876 children (12 studies, 6-12 years, 2014-2021) and 2499 adolescents (10 studies, 12-18 years, 2014-2021) with up to 14 phthalate/DINCH urinary metabolites. We used multilevel linear regression to assess associations between phthalate/DINCH concentrations and BMI z-scores, testing effect modification by sex. In a subset, Bayesian kernel machine regression (BKMR) and quantile-based g-computation assessed important predictors and mixture effects. RESULTS: In children, we found few associations in single pollutant models and no interactions by sex (p-interaction > 0.1). BKMR detected no relevant exposures (posterior inclusion probabilities, PIPs < 0.25), nor joint mixture effect. In adolescent single pollutant analysis, mono-ethyl phthalate (MEP) concentrations were associated with higher BMI z-score in males (ß = 0.08, 95 % CI: 0.001,0.15, per interquartile range increase in ln-transformed concentrations, p-interaction = 0.06). Conversely, mono-isobutyl phthalate (MiBP) was associated with a lower BMI z-score in both sexes (ß = -0.13, 95 % CI: -0.19, -0.07, p-interaction = 0.74), as was sum of di(2-ethylhexyl) phthalate (∑DEHP) metabolites in females only (ß = -0.08, 95 % CI: -0.14, -0.02, p-interaction = 0.01). In BKMR, higher BMI z-scores were predicted by MEP (PIP=0.90) and MBzP (PIP=0.84) in males. Lower BMI z-scores were predicted by MiBP (PIP=0.999), OH-MIDP (PIP=0.88) and OH-MINCH (PIP=0.72) in both sexes, less robustly by DEHP (PIP=0.61) in females. In quantile g-computation, the overall mixture effect was null for males, and trended negative for females (ß = -0.11, 95 % CI: -0.25, 0.03, per joint exposure quantile). CONCLUSION: In this large Europe-wide study, we found age/sex-specific differences between phthalate metabolites and BMI z-score, stronger in adolescents. Longitudinal studies with repeated phthalate measurements are needed.

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.

PFASs: What can we learn from the European Human Biomonitoring Initiative HBM4EU.

Per- and polyfluoroalkyl substances (PFASs) were one of the priority substance groups selected which have been investigated under the ambitious European Joint programme HBM4EU (2017-2022). In order to answer policy relevant questions concerning exposure and health effects of PFASs in Europe several activities were developed under HBM4EU namely i) synthesis of HBM data generated in Europe prior to HBM4EU by developing new platforms, ii) development of a Quality Assurance/Quality Control Program covering 12 biomarkers of PFASs, iii) aligned and harmonized human biomonitoring studies of PFASs. In addition, some cohort studies (on mother-child exposure, occupational exposure to hexavalent chromium) were initiated, and literature researches on risk assessment of mixtures of PFAS, health effects and effect biomarkers were performed. The HBM4EU Aligned Studies have generated internal exposure reference levels for 12 PFASs in 1957 European teenagers aged 12-18 years. The results showed that serum levels of 14.3% of the teenagers exceeded 6.9 µg/L PFASs, which corresponds to the EFSA guideline value for a tolerable weekly intake (TWI) of 4.4 ng/kg for some of the investigated PFASs (PFOA, PFOS, PFNA and PFHxS). In Northern and Western Europe, 24% of teenagers exceeded this level. The most relevant sources of exposure identified were drinking water and some foods (fish, eggs, offal and locally produced foods). HBM4EU occupational studies also revealed very high levels of PFASs exposure in workers (P95: 192 µg/L in chrome plating facilities), highlighting the importance of monitoring PFASs exposure in specific workplaces. In addition, environmental contaminated hotspots causing high exposure to the population were identified. In conclusion, the frequent and high PFASs exposure evidenced by HBM4EU strongly suggests the need to take all possible measures to prevent further contamination of the European population, in addition to adopting remediation measures in hotspot areas, to protect human health and the environment. HBM4EU findings also support the restriction of the whole group of PFASs. Further, research and definition for additional toxicological dose-effect relationship values for more PFASs compounds is needed.

Cross-sectional associations between exposure to per- and polyfluoroalkyl substances and body mass index among European teenagers in the HBM4EU aligned studies.

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 questionnaires. 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 µg/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 [ß-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.

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.

Exposure to flame retardants in European children - Results from the HBM4EU aligned studies.

Many legacy and emerging flame retardants (FRs) have adverse human and environmental health effects. This study reports legacy and emerging FRs in children from nine European countries from the HBM4EU aligned studies. Studies from Belgium, Czech Republic, Germany, Denmark, France, Greece, Slovenia, Slovakia, and Norway conducted between 2014 and 2021 provided data on FRs in blood and urine from 2136 children. All samples were collected and analyzed in alignment with the HBM4EU protocols. Ten halogenated FRs were quantified in blood, and four organophosphate flame retardants (OPFR) metabolites quantified in urine. Hexabromocyclododecane (HBCDD) and decabromodiphenyl ethane (DBDPE) were infrequently detected (<16% of samples). BDE-47 was quantified in blood from Greece, France, and Norway, with France (0.36 ng/g lipid) having the highest concentrations. BDE-153 and -209 were detected in <40% of samples. Dechlorane Plus (DP) was quantified in blood from four countries, with notably high median concentrations of 16 ng/g lipid in Slovenian children. OPFR metabolites had a higher detection frequency than other halogenated FRs. Diphenyl phosphate (DPHP) was quantified in 99% of samples across 8 countries at levels ∼5 times higher than other OPFR metabolites (highest median in Slovenia of 2.43 ng/g lipid). FR concentrations were associated with lifestyle factors such as cleaning frequency, employment status of the father of the household, and renovation status of the house, among others. The concentrations of BDE-47 in children from this study were similar to or lower than FRs found in adult matrices in previous studies, suggesting lower recent exposure and effectiveness of PBDE restrictions.

Time Patterns in Internal Human Exposure Data to Bisphenols, Phthalates, DINCH, Organophosphate Flame Retardants, Cadmium and Polyaromatic Hydrocarbons in Europe.

Human biomonitoring (HBM) data in Europe are often fragmented and collected in different EU countries and sampling periods. Exposure levels for children and adult women in Europe were evaluated over time. For the period 2000-2010, literature and aggregated data were collected in a harmonized way across studies. Between 2011-2012, biobanked samples from the DEMOCOPHES project were used. For 2014-2021, HBM data were generated within the HBM4EU Aligned Studies. Time patterns on internal exposure were evaluated visually and statistically using the 50th and 90th percentiles (P50/P90) for phthalates/DINCH and organophosphorus flame retardants (OPFRs) in children (5-12 years), and cadmium, bisphenols and polycyclic aromatic hydrocarbons (PAHs) in women (24-52 years). Restricted phthalate metabolites show decreasing patterns for children. Phthalate substitute, DINCH, shows a non-significant increasing pattern. For OPFRs, no trends were statistically significant. For women, BPA shows a clear decreasing pattern, while substitutes BPF and BPS show an increasing pattern coinciding with the BPA restrictions introduced. No clear patterns are observed for PAHs or cadmium. Although the causal relations were not studied as such, exposure levels to chemicals restricted at EU level visually decreased, while the levels for some of their substitutes increased. The results support policy efficacy monitoring and the policy-supportive role played by HBM.

From science to policy: How European HBM indicators help to answer policy questions related to phthalates and DINCH exposure.

Within the European Human Biomonitoring (HBM) Initiative HBM4EU we derived HBM indicators that were designed to help answering key policy questions and support chemical policies. The result indicators convey information on chemicals exposure of different age groups, sexes, geographical regions and time points by comparing median exposure values. If differences are observed for one group or the other, policy measures or risk management options can be implemented. Impact indicators support health risk assessment by comparing exposure values with health-based guidance values, such as human biomonitoring guidance values (HBM-GVs). In general, the indicators should be designed to translate complex scientific information into short and clear messages and make it accessible to policy makers but also to a broader audience such as stakeholders (e.g. NGO's), other scientists and the general public. Based on harmonized data from the HBM4EU Aligned Studies (2014-2021), the usefulness of our indicators was demonstrated for the age group children (6-11 years), using two case examples: one phthalate (Diisobutyl phthalate: DiBP) and one non-phthalate substitute (Di-isononyl cyclohexane-1,2- dicarboxylate: DINCH). For the comparison of age groups, these were compared to data for teenagers (12-18 years), and time periods were compared using data from the DEMOCOPHES project (2011-2012). Our result indicators proved to be suitable for demonstrating the effectiveness of policy measures for DiBP and the need of continuous monitoring for DINCH. They showed similar exposure for boys and girls, indicating that there is no need for gender focused interventions and/or no indication of sex-specific exposure patterns. They created a basis for a targeted approach by highlighting relevant geographical differences in internal exposure. An adequate data basis is essential for revealing differences for all indicators. This was particularly evident in our studies on the indicators on age differences. The impact indicator revealed that health risks based on exposure to DiBP cannot be excluded. This is an indication or flag for risk managers and policy makers that exposure to DiBP still is a relevant health issue. HBM indicators derived within HBM4EU are a valuable and important complement to existing indicator lists in the context of environment and health. Their applicability, current shortcomings and solution strategies are outlined.

Current exposure to phthalates and DINCH in European children and adolescents - Results from the HBM4EU Aligned Studies 2014 to 2021.

Phthalates are mainly used as plasticizers for polyvinyl chloride (PVC). Exposure to several phthalates is associated with different adverse effects most prominently on the development of reproductive functions. The HBM4EU Aligned Studies (2014-2021) have investigated current European exposure to ten phthalates (DEP, BBzP, DiBP, DnBP, DCHP, DnPeP, DEHP, DiNP, DiDP, DnOP) and the substitute DINCH to answer the open policy relevant questions which were defined by HBM4EU partner countries and EU institutions as the starting point of the programme. The exposure dataset includes ∼5,600 children (6-11 years) and adolescents (12-18 years) from up to 12 countries per age group and covering the North, East, South and West European regions. Study data from participating studies were harmonised with respect to sample size and selection of participants, selection of biomarkers, and quality and comparability of analytical results to provide a comparable perspective of European exposure. Phthalate and DINCH exposure were deduced from urinary excretions of metabolites, where concentrations were expressed as their key descriptor geometric mean (GM) and 95th percentile (P95). This study aims at reporting current exposure levels and differences in these between European studies and regions, as well as comparisons to human biomonitoring guidance values (HBM-GVs). GMs for children were highest for ∑DEHP metabolites (33.6 µg/L), MiBP (26.6 µg/L), and MEP (24.4 µg/L) and lowest for∑DiDP metabolites (1.91 µg/L) and ∑DINCH metabolites (3.57 µg/L). In adolescents highest GMs were found for MEP (43.3 µg/L), ∑DEHP metabolites (28.8 µg/L), and MiBP (25.6 µg/L) and lowest for ∑DiDP metabolites (= 2.02 µg/L) and ∑DINCH metabolites (2.51 µg/L). In addition, GMs and P95 stratified by European region, sex, household education level, and degree of urbanization are presented. Differences in average biomarker concentrations between sampling sites (data collections) ranged from factor 2 to 9. Compared to the European average, children in the sampling sites OCC (Denmark), InAirQ (Hungary), and SPECIMEn (The Netherlands) had the lowest concentrations across all metabolites and ESTEBAN (France), NAC II (Italy), and CROME (Greece) the highest. For adolescents, comparably higher metabolite concentrations were found in NEB II (Norway), PCB cohort (Slovakia), and ESTEBAN (France), and lower concentrations in POLAES (Poland), FLEHS IV (Belgium), and GerES V-sub (Germany). Multivariate analyses (Survey Generalized Linear Models) indicate compound-specific differences in average metabolite concentrations between the four European regions. Comparison of individual levels with HBM-GVs revealed highest rates of exceedances for DnBP and DiBP, with up to 3 and 5%, respectively, in children and adolescents. No exceedances were observed for DEP and DINCH. With our results we provide current, detailed, and comparable data on exposure to phthalates in children and - for the first time - in adolescents, and - for the first time - on DINCH in children and adolescents of all four regions of Europe which are particularly suited to inform exposure and risk assessment and answer open policy relevant questions.

Gene expression signatures in PCB-exposed Slovak children in relation to their environmental exposures and socio-physical characteristics.

Our previous gene expression studies in a PCB-exposed cohort of young children in Slovakia revealed that early-life exposures to PCBs and other organochlorine compounds were associated with significant alterations across several pathogenetic pathways. The present study was undertaken to further explore the high-throughput qRT-PCR-based gene expression effects by using TaqMan low-density array (TLDA) for selected genes in a sample of 55 children from the cohort. We analyzed the transcriptional changes of 11 genes in relation to PCB and organochlorine pesticide exposure levels (including DDT, DDE, HCH, and HCB), and to BMI and ethnicity in this cohort. The results indicated an overall downregulation of expression of these genes. Maximum downregulation (in fold change) was observed in the ENTPD3 gene, and the minimum level of downregulation was in CYP2D6. As per our multinomial regression model study, downregulation of LEPR gene was significantly directly correlated with all the exposure variables. Downregulation of APC, ARNT, CYP2D6, LEPR, LRP12, and MYC genes was directly correlated with BMI (kg/m2) of the individuals. Gender-specific differences in gene expression were observed in CYP2D6 (p-value 0.0001) and LEPR (p-value 0.028), while downregulation of CYP2D6 (p-value 0.01), LEPR (p-value 0.02), LRP12 (p-value 0.04), and MYC (p-value 0.02) genes was consistently observed in Roma children compared to Caucasians. The investigation of such health disparities must be emphasized in future research, together with interventions to reduce the health consequences of PCB exposures. In this context, we emphasize the importance of biomarker-based approaches to future research on genetic susceptibility to the effects of these compounds.