Early heart and skeletal muscle mitochondrial response to a moderate hypobaric hypoxia environment.

In eukaryotic cells, aerobic energy is produced by mitochondria through oxygen uptake. However, little is known about the early mitochondrial responses to moderate hypobaric hypoxia (MHH) in highly metabolic active tissues. Here, we describe the mitochondrial responses to acute MHH in the heart and skeletal muscle. Rats were randomly allocated into a normoxia control group (n = 10) and a hypoxia group (n = 30), divided into three groups (0, 6, and 24 h post-MHH). The normoxia situation was recapitulated at the University of Granada, at 662 m above sea level. The MHH situation was performed at the High-Performance Altitude Training Centre of Sierra Nevada located in Granada at 2320 m above sea level. We found a significant increase in mitochondrial supercomplex assembly in the heart as soon as the animals reached 2320 m above sea level and their levels are maintained 24 h post-exposure, but not in skeletal muscle. Furthermore, in skeletal muscle, at 0 and 6 h, there was increased dynamin-related protein 1 (Drp1) expression and a significant reduction in Mitofusin 2. In conclusion, mitochondria from the muscle and heart respond differently to MHH: mitochondrial supercomplexes increase in the heart, whereas, in skeletal muscle, the mitochondrial pro-fission response is trigged. Considering that skeletal muscle was not actively involved in the ascent when the heart was beating faster to compensate for the hypobaric, hypoxic conditions, we speculate that the different responses to MHH are a result of the different energetic requirements of the tissues upon MHH. KEY POINTS: The heart and the skeletal muscle showed different mitochondrial responses to moderate hypobaric hypoxia. Moderate hypobaric hypoxia increases the assembly of the electron transport chain complexes into supercomplexes in the heart. Skeletal muscle shows an early mitochondrial pro-fission response following exposure to moderate hypobaric hypoxia.

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.

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.

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.

Xeno-estrogenic activity of real-life mixtures of perfluoroalkylated substances in human placenta homogenates.

Humans are simultaneously exposed to complex chemical mixtures, and its combined effect can affect human health. As part of the HBM4EU project, the actual mixture of perfluoroalkylated substances (PFAS) in 25 human placenta samples was extracted by chromatographic methods and assessed for xeno-estrogenic activity using two in-vitro bioassays: the estrogen receptor transactivity and the E-Screen assay. Most of the PFAS extracts displayed xeno-estrogenic activity, in one or both assays. The xeno-estrogenic activities in the two bioassays were not correlated, but both assays showed an overall negative correlation with placenta concentrations of single PFAS. Xeno-estrogenic activities were significantly related to maternal characteristics; being higher in young, smokers and primiparous women, but not with fetal growth (birth weight, birth length, head circumference, gestational age, placenta weight). The presented extraction method can be used to study the combined effect of real-life mixtures of PFAS in relation to health outcomes in large-scale human biomonitoring studies.

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.

Rapid extraction and analysis of oxidative stress and DNA damage biomarker 8-hydroxy-2'-deoxyguanosine (8-OHdG) in urine: Application to a study with pregnant women.

Oxidative stress is an important toxicity and genotoxicity mechanism of many chronic adverse health outcomes. 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 creatinine-adjusted 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.

Implementation of effect biomarkers in human biomonitoring studies: A systematic approach synergizing toxicological and epidemiological knowledge.

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 validated 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 toxicological 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 confirms 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 understand the public health implications of human exposure to environmental chemicals.

Human risk associated with exposure to mixtures of antiandrogenic chemicals evaluated using in vitro hazard and human biomonitoring data.

BACKGROUND: Scientific evidence for underestimated toxicity from unintentional exposure to chemical mixtures is mounting. Yet, harmonized approaches on how to assess the actual risk of mixtures is lacking. As part of the European Joint programme 'Human Biomonitoring for Europe' we explored a novel methodology for mixture risk assessment of chemicals affecting male reproductive function. METHODOLOGY: We explored a methodology for chemical mixture risk assessment based on human in vitro data combined with human exposure data, thereby circumventing the drawbacks of using hazard data from rodents and estimated exposure intake levels. Human androgen receptor (hAR) antagonism was selected as the most important molecular initiating event linked to adverse outcomes on male reproductive health. RESULTS: Our work identified 231 chemicals able to interfere with hAR activity. Among these were 61 finally identified as having both reliable hAR antagonist and human biomonitoring data. Calculation of risk quotients indicated that PCBs (118, 138, 157), phthalates (BBP, DBP, DIBP), benzophenone-3, PFOS, methylparaben, triclosan, some pesticides (i.e cypermethrin, ß-endosulfan, methylparathion, p,p-DDE), and a PAH metabolite (1-hydroxypyrene) contributed to the mixture effect. The major chemical mixture drivers were PCB 118, BBP, PFOS, DBP, and the UV filter benzophenone-3, together contributing with 75% of the total mixture effect that was primarily driven by high exposure values. CONCLUSIONS: This viable way forward for mixture risk assessment of chemicals has the advantages of (1) being a more comprehensive mixture risk assessment also covering data-poor chemicals, and (2) including human data only. However, the approach is subjected to uncertainties in terms of in vitro to in vivo extrapolation, it is not ready for decision making, and needs further development. Still, the results indicate a concern for adverse effects on reproductive function in highly exposed boys, especially when considering additional exposure to data-poor chemicals and chemicals acting by other mechanisms of action.

Kisspeptin as potential biomarker of environmental chemical mixture effect on reproductive hormone profile: A pilot study in adolescent males.

BACKGROUND: Kisspeptin has been proposed as an effect biomarker to understand the mechanisms by which some environmental chemicals adversely affect the human reproductive system. OBJECTIVE: To ascertain whether kisspeptin serum protein and DNA methylation levels are associated with exposure to several environmental chemicals (individually and as a mixture) and serum reproductive hormone levels in adolescent males. METHODS: Three phenols (bisphenol A [BPA], methyl-paraben [MPB], and benzophenone-3 [BP3]); two toxic metals (arsenic and cadmium); and four metabolites of non-persistent pesticides, including insecticides (2-isopropyl-6-methyl-4-pyrimidinol [IMPy], malathion diacid [MDA], and dimethylcyclopropane carboxylic acid [DCCA]) and fungicides (ethylene thiourea [ETU]) were measured in first-morning urine samples of 133 adolescent males aged 15-17 years from the INMA-Granada cohort. In blood samples collected on the same day, KISS1 gene DNA methylation was measured at four CpGs from the Exon IV, as well as serum levels of kiss54 protein, total testosterone (T), estradiol (E2), sex hormone binding-globulin, dehydroepiandrosterone sulfate, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). Multiple linear regression and mixture (quantile g-computation) models were fit. RESULTS: Urinary MDA and DCCA concentrations were associated with higher kiss54 levels [% change (95%CI) for each log-unit increase in concentration = 2.90 (0.32;5.56), and 1.93 (0.45,3.43), respectively]; IMPy with lower DNA methylation percentage at CpG1 and total CpGs [% change (95%CI) = -1.15 (-1.96;-0.33): -0.89 (-1.73;-0.01), respectively]; and BP3 and DCCA with lower total CpGs methylation [-0.53 (-1.04;-0.01) and - 0.69 (-1.37;-0.01), respectively]. The pesticide mixture and the whole chemical mixture were associated with higher kiss54 [% change (95%CI) = 9.09 (3.29;15.21) and 11.61 (3.96;19.82), respectively] and lower methylation levels at several CpGs. Additionally, serum kiss54 in the third tertile was associated with higher LH levels [% change (95%CI) = 28.69 (3.75-59.63)], and third-tertile CpG1, CpG2, and total CpG methylation percentages were associated with lower FSH and E2. CONCLUSION: The findings of the present study and the negative correlation between serum kiss54 levels and KISS1 DNA methylation percentages suggested that kisspeptin may be a promising effect biomarker.

Brain-derived neurotrophic factor (BDNF): an effect biomarker of neurodevelopment in human biomonitoring programs.

The present narrative review summarizes recent findings focusing on the role of brain-derived neurotrophic factor (BDNF) as a biomarker of effect for neurodevelopmental alterations during adolescence, based on health effects of exposure to environmental chemical pollutants. To this end, information was gathered from the PubMed database and the results obtained in the European project Human Biomonitoring for Europe (HBM4EU), in which BDNF was measured at two levels of biological organization: total BDNF protein (serum) and BDNF gene DNA methylation (whole blood) levels. The obtained information is organized as follows. First, human biomonitoring, biomarkers of effect and the current state of the art on neurodevelopmental alterations in the population are presented. Second, BDNF secretion and mechanisms of action are briefly explained. Third, previous studies using BDNF as an effect biomarker were consulted in PubMed database and summarized. Finally, the impact of bisphenol A (BPA), metals, and non-persistent pesticide metabolites on BDNF secretion patterns and its mediation role with behavioral outcomes are addressed and discussed. These findings were obtained from three pilot studies conducted in HBM4EU project. Published findings suggested that exposure to some chemical pollutants such as fine particle matter (PM), PFAS, heavy metals, bisphenols, and non-persistent pesticides may alter circulating BDNF levels in healthy population. Therefore, BDNF could be used as a valuable effect biomarker to investigate developmental neurotoxicity of some chemical pollutants.

Genome-wide distribution of histone trimethylation reveals a global impact of bisphenol A on telomeric binding proteins and histone acetyltransferase factors: a pilot study with human and in vitro data.

OBJECTIVE: To assess the genetic and epigenetic effects promoted by Bisphenol A (BPA) exposure in adolescent males from the Spanish INMA-Granada birth cohort, and 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). Analyses were performed with material extracted from whole blood of the adolescents, complemented by in vitro assessments of human (HeLa) cells exposed to 10 nM BPA, specifically, immunofluorescence evaluation of protein levels, gene expression analysis and ChIP‒qPCR analysis. RESULTS: Adolescents in the high urinary BPA levels group presented a higher level of Satellite A (SATA) repetitive region copy numbers compared to those in the low BPA group and a tendency towards increase in telomere length. We also observed decreased DNA methylation at the promoters of the imprinted genes H19, KCNQ1, and IGF2; at LINE1 retroelements; and at the ARID2, EGFR and ESRRA and TERT genes. Genome-wide sequencing revealed increased H3K4me3 occupancy at the promoters of genes encoding histone acetyltransferases, telomeric DNA binding factors and DNA repair genes. Results were supported in HeLa cells exposed to 10 nM BPA in vitro. In accordance with the data obtained in blood samples, we observed higher H3K4me3 occupancy and lower DNA methylation at some specific targets in HeLa cells. In exposed cells, changes in the expression of genes encoding DNA repair factors (ATM, ARID2, TRP53) were observed, and increased expression of several genes encoding telomeric DNA binding factors (SMG7, TERT, TEN1, UPF1, ZBTB48) were also found. Furthermore, an increase in ESR1/ERa was observed in the nuclei of HeLa cells along with increased binding of ESR1 to KAT5, KMT2E and TERF2IP promoters and decreased ESR1 binding at the RARA promoter. The DNA damage marker p53/TP53 was also increased. CONCLUSION: In this pilot study, genome-wide analysis of histone trimethylation in adolescent males exposed to BPA revealed a global impact on the expression of genes encoding telomeric binding proteins and histone acetyltransferase factors with similar results in HeLa cells. Nevertheless, larger studies should confirm our findings.

Exposure to non-persistent pesticides, BDNF, and behavioral function in adolescent males: Exploring a novel effect biomarker approach.

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.

Exploring the relationship between metal exposure, BDNF, and behavior in adolescent males.

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.

BDNF as a potential mediator between childhood BPA exposure and behavioral function in adolescent boys from the INMA-Granada cohort.

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 (ß = 0.76; 95% CI: 0.02, 1.49) and somatic complaints (ß = 0.80; 95% CI: -0.16, 1.75) at adolescence. BPA concentrations were positively associated with BDNF DNA methylation at CpG6 (ß = 0.21; 95% CI: 0.06, 0.36) and mean CpG methylation (ß = 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 ≤ 0.01), and CpG6-thought problems (p-trend ≤ 0.01). A significant mediated effect by CpG6 DNA methylation was observed (ß = 0.23; 95% CI: 0.01, 0.57), accounting for up to 34% of the BPA-thought problems association. CONCLUSIONS: In line with toxicological studies, BPA exposure was longitudinally associated with increased BDNF DNA methylation, supporting the biological plausibility of BPA-behavior relationships previously described in the epidemiological literature. Given its novelty and preliminary nature, this effect biomarker approach should be replicated in larger birth cohorts.

Development and validation of brain-derived neurotrophic factor measurement in human urine samples as a non-invasive effect biomarker.

Background: Brain-derived neurotrophic factor (BDNF), a neurotrophic growth factor mainly expressed in the brain, has been proposed as a potential effect biomarker; that is, as a measurable biomarker whose values could be associated with several diseases, including neurological impairments. The European Human Biomonitoring Initiative (HBM4EU) has also recognized effect biomarkers as a useful tool for establishing link between exposure to environmental pollutants and human health. Despite the well-establish protocol for measuring serum BDNF, there is a need to validate its assessment in urine, a non-invasive sample that can be easily repeated over time. The aim of this study was to develop, standardize and validate a methodology to quantify BDNF protein levels in urine samples before its implementation in biomonitoring studies. Methods: Different experimental conditions and non-competitive commercial enzyme-linked immunosorbent assay (ELISA) kits were tested to determine the optimal analytical procedure, trying to minimize the shortcomings of ELISA kits. The fine-tune protocol was validated in a pilot study using both upon awakening (n = 150) and prior to sleeping (n = 106) urine samples from the same Spanish adolescent males in a well-characterized study population (the Spanish INMA-Granada cohort). Results: The best results were obtained in 0.6 ml of urine after the acidification and extraction (pre-concentration) of samples. The highest reproducibility was obtained with the ELISA kit from Raybiotech. Urinary BDNF concentrations of adolescent males were within the previously reported range (morning = 0.047-6.801 ng/ml and night = 0.047-7.404 ng/ml). Urinary BDNF levels in the awakening and pre-sleep samples did not follow a normal distribution and were not correlated. Conclusion: The developed methodology offers good sensitivity and reproducibility. Having reliable markers in urine may facilitate both diagnosis and monitoring possible diseases (and treatment). Further studies are needed to implement urinary BDNF in biomonitoring studies to further elucidate its usefulness and biological significance for neurological impairments.

Assessment of chemical mixtures using biomarkers of combined biological activity: A screening study in human placentas.

Humans are simultaneously exposed to complex mixtures of chemicals with limited knowledge on potential health effects, therefore improved tools for assessing these mixtures are needed. As part of the Human Biomonitoring for Europe (HBM4EU) Project, we aimed to examine the combined biological activity of chemical mixtures extracted from human placentas using one in vivo and four in vitro bioassays, also known as biomarkers of combined effect. Relevant endocrine activities (proliferative and/or reporter gene assays) and four endpoints were tested: the estrogen receptor (ER), androgen receptor (AR), and aryl hydrocarbon receptor (AhR) activities, as well as thyroid hormone (TH) signaling. Correlations among bioassays and their functional shapes were evaluated. Results showed that all placental extracts agonized or antagonized at least three of the abovementioned endpoints. Most placentas induced ER-mediated transactivation and ER-dependent cell proliferation, together with a strong inhibition of TH signaling and the AR transactivity; while the induction of the AhR was found in only one placental extract. The effects in the two estrogenic bioassays were positively and significantly correlated and the AR-antagonism activity showed a positive borderline-significant correlation with both estrogenic bioassay activities. However, the in vivo anti-thyroid activities of placental extracts were not correlated with any of the tested in vitro assays. Findings highlight the importance of comprehensively mapping the biological effects of "real-world" chemical mixtures present in human samples, through a battery of in vitro and in vivo bioassays. This approach should be a complementary tool for epidemiological studies to further elucidate the combined biological fingerprint triggered by chemical mixtures.

Receptor-based in vitro activities to assess human exposure to chemical mixtures and related health impacts.

Humans are exposed to a large number of chemicals from sources such as the environment, food, and consumer products. There is growing concern that human exposure to chemical mixtures, especially during critical periods of development, increases the risk of adverse health effects in newborns or later in life. Historically, the one-chemical-at-a-time approach has been applied both for exposure assessment and hazard characterisation, leading to insufficient knowledge about human health effects caused by exposure to mixtures of chemicals that have the same target. To circumvent this challenge researchers can apply in vitro assays to analyse both exposure to and human health effects of chemical mixtures in biological samples. The advantages of using in vitro assays are: (i) that an integrated effect is measured, taking combined mixture effects into account and (ii) that in vitro assays can reduce complexity in identification of Chemicals of Emerging Concern (CECs) in human tissues. We have reviewed the state-of-the-art on the use of receptor-based in vitro assays to assess human exposure to chemical mixtures and related health impacts. A total of 43 studies were identified, in which endpoints for the arylhydrocarbon receptor (AhR), the estrogen receptor (ER), and the androgen receptor (AR) were used. The majority of studies reported biological activities that could be associated with breast cancer incidence, male reproductive health effects, developmental toxicities, human demographic characteristics or lifestyle factors such as dietary patterns. A few studies used the bioactivities to check the coverage of the chemical analyses of the human samples, whereas in vitro assays have so far not regularly been used for identifying CECs in human samples, but rather in environmental matrices or food packaging materials. A huge field of novel applications using receptor-based in vitro assays for mixture toxicity assessment on human samples and effect-directed analysis (EDA) using high resolution mass spectrometry (HRMS) for identification of toxic compounds waits for exploration. In the future this could lead to a paradigm shift in the way we unravel adverse human health effects caused by chemical mixtures.

Bisphenol A and its analogues: A comprehensive review to identify and prioritize effect biomarkers for human biomonitoring.

Human biomonitoring (HBM) studies have demonstrated widespread and daily exposure to bisphenol A (BPA). Moreover, BPA structural analogues (e.g. BPS, BPF, BPAF), used as BPA replacements, are being increasingly detected in human biological matrices. BPA and some of its analogues are classified as endocrine disruptors suspected of contributing to adverse health outcomes such as altered reproduction and neurodevelopment, obesity, and metabolic disorders among other developmental and chronic impairments. One of the aims of the H2020 European Human Biomonitoring Initiative (HBM4EU) is the implementation of effect biomarkers at large scales in future HBM studies in a systematic and standardized way, in order to complement exposure data with mechanistically-based biomarkers of early adverse effects. This review aimed to identify and prioritize existing biomarkers of effect for BPA, as well as to provide relevant mechanistic and adverse outcome pathway (AOP) information in order to cover knowledge gaps and better interpret effect biomarker data. A comprehensive literature search was performed in PubMed to identify all the epidemiologic studies published in the last 10 years addressing the potential relationship between bisphenols exposure and alterations in biological parameters. A total of 5716 references were screened, out of which, 119 full-text articles were analyzed and tabulated in detail. This work provides first an overview of all epigenetics, gene transcription, oxidative stress, reproductive, glucocorticoid and thyroid hormones, metabolic and allergy/immune biomarkers previously studied. Then, promising effect biomarkers related to altered neurodevelopmental and reproductive outcomes including brain-derived neurotrophic factor (BDNF), kisspeptin (KiSS), and gene expression of nuclear receptors are prioritized, providing mechanistic insights based on in vitro, animal studies and AOP information. Finally, the potential of omics technologies for biomarker discovery and its implications for risk assessment are discussed. To the best of our knowledge, this is the first effort to comprehensively identify bisphenol-related biomarkers of effect for HBM purposes.

Influence of a Multidisciplinary Program of Diet, Exercise, and Mindfulness on the Quality of Life of Stage IIA-IIB Breast Cancer Survivors.

Background: Integrative oncology has proven to be a useful approach to control cancer symptoms and improve the quality of life (QoL) and overall health of patients, delivering integrated patient care at both physical and emotional levels. The objective of this randomized trial was to evaluate the effects of a triple intervention program on the QoL and lifestyle of women with breast cancer. Methods: Seventy-five survivors of stage IIA-IIB breast cancer were randomized into 2 groups. The intervention group (IG) received a 6-month dietary, exercise, and mindfulness program that was not offered to the control group (CG). Data were gathered at baseline and at 6 months postintervention on QoL and adherence to Mediterranean diet using clinical markers and validated questionnaires. Between-group differences at baseline and 3 months postintervention were analyzed using Student's t test for related samples and the Wilcoxon and Mann-Whitney U tests. Results: At 6 months postintervention, the IG showed significant improvements versus CG in physical functioning (p = .027), role functioning (p = .028), and Mediterranean diet adherence (p = .02) and a significant reduction in body mass index (p = .04) and weight (p = .05), with a mean weight loss of 0.7 kg versus a gain of 0.55 kg by the CG (p = .05). Dyspnea symptoms were also increased in the CG versus IG (p = .066). Conclusions: These results demonstrate that an integrative dietary, physical activity, and mindfulness program enhances the QoL and healthy lifestyle of stage IIA-IIB breast cancer survivors. Cancer symptoms may be better managed by the implementation of multimodal rather than isolated interventions.