Environmental Exposure to Persistent Organic Pollutants and Its Association with Endometriosis Risk: Implications in the Epithelial-Mesenchymal Transition Process.

We aimed to explore the relationship of adipose tissue concentrations of some persistent organic pollutants (POPs) with the risk of endometriosis and the endometriotic tissue expression profile of genes related to the endometriosis-related epithelial-mesenchymal transition (EMT) process. This case-control study enrolled 109 women (34 cases and 75 controls) between January 2018 and March 2020. Adipose tissue samples and endometriotic tissues were intraoperatively collected to determine concentrations of nine POPs and the gene expression profiles of 36 EMT-related genes, respectively. Associations of POPs with endometriosis risk were explored with multivariate logistic regression, while the relationship between exposure and gene expression profiles was assessed through Spearman correlation or Mann-Whitney U tests. After adjustment, increased endometriosis risk was associated with p,p'-DDT, PCB-180, and ΣPCBs. POP exposure was also associated with reduced gene expression levels of the CLDN7 epithelial marker and increased levels of the ITGB2 mesenchymal marker and a variety of EMT promoters (HMGA1, HOXA10, FOXM1, DKK1, CCR1, TNFRSF1B, RRM2, ANG, ANGPT1, and ESR1). Our findings indicate that exposure to POPs may increase the risk of endometriosis and might have a role in the endometriosis-related EMT development, contributing to the disease onset and progression. Further studies are warranted to corroborate these findings.

Expression Profiles of Genes Related to Development and Progression of Endometriosis and Their Association with Paraben and Benzophenone Exposure.

Increasing evidence has been published over recent years on the implication of endocrine-disrupting chemicals (EDCs), including parabens and benzophenones in the pathogenesis and pathophysiology of endometriosis. However, to the best of our knowledge, no study has been published on the ways in which exposure to EDCs might affect cell-signaling pathways related to endometriosis. We aimed to describe the endometriotic tissue expression profile of a panel of 23 genes related to crucial cell-signaling pathways for the development and progression of endometriosis (cell adhesion, invasion/migration, inflammation, angiogenesis, and cell proliferation/hormone stimulation) and explore its relationship with the exposure of patients to parabens (PBs) and benzophenones (BPs). This cross-sectional study included a subsample of 33 women with endometriosis from the EndEA study, measuring their endometriotic tissue expressions of 23 genes, while urinary concentrations of methyl-, ethyl-, propyl-, butyl-paraben, benzophenone-1, benzophenone-3, and 4-hydroxybenzophenone were determined in 22 women. Spearman's correlations test and linear and logistic regression analyses were performed. The expression of 52.2% of studied genes was observed in >75% of endometriotic tissue samples and the expression of 17.4% (n = 4) of them in 50-75%. Exposure to certain PB and BP congeners was positively associated with the expression of key genes for the development and proliferation of endometriosis. Genes related to the development and progression of endometriosis were expressed in most endometriotic tissue samples studied, suggesting that exposure of women to PBs and BPs may be associated with the altered expression profile of genes related to cellular pathways involved in the development of endometriosis.

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.

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.

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.

The Mixture of Bisphenol-A and Its Substitutes Bisphenol-S and Bisphenol-F Exerts Obesogenic Activity on Human Adipose-Derived Stem Cells.

Bisphenol A (BPA) and its substitutes, bisphenol F (BPF) and S (BPS), have previously shown in vitro obesogenic activity. This study was designed to investigate their combined effect on the adipogenic differentiation of human adipose-derived stem cells (hASCs). Cells were exposed for 14 days to an equimolar mixture of bisphenols (MIX) (range 10 nM-10 µM). Oil Red staining was used to measure intracellular lipid accumulation, quantitative real-time polymerase chain reaction (qRT-PCR) to study gene expression of adipogenic markers (PPARγ, C/EBPα, LPL, and FABP4), and Western Blot to determine their corresponding proteins. The MIX promoted intracellular lipid accumulation in a dose-dependent manner with a maximal response at 10 µM. Co-incubation with pure antiestrogen (ICI 182,780) inhibited lipid accumulation, suggesting that the effect was mediated by the estrogen receptor. The MIX also significantly altered the expression of PPARγ, C/EBPα, LPL, and FABP4 markers, observing a non-monotonic (U-shaped) dose-response, with maximal gene expression at 10 nM and 10 µM and lesser expression at 1 µM. This pattern was not observed when bisphenols were tested individually. Exposure to MIX (1-10 µM) also increased all encoded proteins except for FABP4, which showed no changes. Evaluation of the combined effect of relevant chemical mixtures is needed rather than single chemical testing.

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.

Bisphenol F and bisphenol S promote lipid accumulation and adipogenesis in human adipose-derived stem cells.

Bisphenol F (BPF) and bisphenol S (BPS) are increasingly used as substitutes for bisphenol A (BPA), an endocrine disrupting chemical (EDC) with obesogenic activity. We investigated the in vitro effects of BPS and BPF on the adipogenesis of human adipose-derived stem cells (hASCs) exposed to different doses (0.01, 0.1, 1, 10 and 25 µM), stopping the adipogenic process at 7 or 14 days. Intracellular lipid accumulation was quantified by the Oil Red O assay, gene expression of peroxisome proliferator-activated receptor gamma (PPARγ), CCAT/enhancer-binding protein (C/EBPα), lipoprotein-lipase (LPL) and fatty acid binding protein 4 (FABP4), by quantitative real-time polymerase chain reaction (qRT-PCR) and protein levels by Western Blot. hASCs with BPF or BPS produced a linear dose-response increase in intracellular lipid accumulation and in gene expression of the adipogenic markers, confirmed by protein levels. Co-treatment ICI 182,780 significantly inhibited BPF- but not BPS-induced lipid accumulation. Given the affinity of bisphenols for diverse nuclear receptors, their obesogenic effects may result from a combination of pathways rather than a single mechanism. Further research is warranted on the manner in which chemicals interfere with adipogenic differentiation. To our best knowledge, this report shows for the first time the obesogenic potential of BPF in hASCs.

Organophosphate pesticide exposure, hormone levels, and interaction with PON1 polymorphisms in male adolescents.

OBJECTIVE: To examine the association between urinary metabolites of organophosphate (OP) pesticides and serum concentrations of thyroid and reproductive hormones in male adolescents and to assess the potential effect of interactions between OP pesticides and paraoxonase 1 (PON1) polymorphisms on hormone levels. METHODS: Study subjects (N = 117) were male 16- to 17-year-olds from the Environment and Childhood (INMA)-Granada cohort in Spain. Concentrations of 3,5,6-trichloro-2-pyridinol (TCPy), a metabolite of chlorpyrifos/chlorpyrifos-methyl, 2-isopropyl-6-methyl-4-pyrimidinol (IMPy), a metabolite of diazinon, and diethylthiophosphate (DETP) and diethyldithiophosphate (DEDTP), non-specific metabolites of OP pesticides, were measured in a spot urine sample from each subject and adjusted for creatinine. Levels of reproductive hormones (total testosterone [TT], estradiol [E2], dehydroepiandrosterone sulfate [DHEAS], sex hormone binding globulin [SHBG], luteinizing hormone [LH], follicle stimulating hormone [FSH], anti-Müllerian hormone [AMH], insulin growth factor 1 [IGF-1], and prolactin), thyroid hormones (free thyroxine [FT4], total triiodothyronine [TT3], and thyroid stimulating hormone [TSH]), and PON1 Q192R and L55M polymorphisms were determined in blood drawn during the same clinical visit. RESULTS: Multiple linear regression models showed that detectable levels of TCPy were associated with an increase in DHEAS and decreases in E2, FSH, and AMH; detectable IMPy with increases in E2, DHEAS, FSH, AMH, and prolactin and decreases in SHBG and LH; and detectable DETP with marginally-significant increases in TT and TT3 and decreases in FSH, AMH, and prolactin. The effect of IMPy and DETP on DHEAS and TT levels, respectively, was higher in subjects that carried the PON1 55MM genotype, while the effect of TCPy, IMPy, and DETP on thyroid hormone levels was higher in PON1 192QR/RR or 55MM genotype carriers. CONCLUSIONS: In male adolescents, non-occupational exposure to OP pesticides was associated with several changes in reproductive and thyroid hormone levels, and the magnitude of some associations was greater in adolescents genetically more susceptible to OP pesticide exposure who carry the PON1 55MM genotype.