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.

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.

Minicells as an Escherichia coli mechanism for the accumulation and disposal of fluorescent cadmium sulphide nanoparticles.

BACKGROUND: Bacterial biosynthesis of fluorescent nanoparticles or quantum dots (QDs) has emerged as a unique mechanism for heavy metal tolerance. However, the physiological pathways governing the removal of QDs from bacterial cells remains elusive. This study investigates the role of minicells, previously identified as a means of eliminating damaged proteins and enhancing bacterial resistance to stress. Building on our prior work, which unveiled the formation of minicells during cadmium QDs biosynthesis in Escherichia coli, we hypothesize that minicells serve as a mechanism for the accumulation and detoxification of QDs in bacterial cells. RESULTS: Intracellular biosynthesis of CdS QDs was performed in E. coli mutants ΔminC and ΔminCDE, known for their minicell-producing capabilities. Fluorescence microscopy analysis demonstrated that the generated minicells exhibited fluorescence emission, indicative of QD loading. Transmission electron microscopy (TEM) confirmed the presence of nanoparticles in minicells, while energy dispersive spectroscopy (EDS) revealed the coexistence of cadmium and sulfur. Cadmium quantification through flame atomic absorption spectrometry (FAAS) demonstrated that minicells accumulated a higher cadmium content compared to rod cells. Moreover, fluorescence intensity analysis suggested that minicells accumulated a greater quantity of fluorescent nanoparticles, underscoring their efficacy in QD removal. Biosynthesis dynamics in minicell-producing strains indicated that biosynthesized QDs maintained high fluorescence intensity even during prolonged biosynthesis times, suggesting continuous QD clearance in minicells. CONCLUSIONS: These findings support a model wherein E. coli utilizes minicells for the accumulation and removal of nanoparticles, highlighting their physiological role in eliminating harmful elements and maintaining cellular fitness. Additionally, this biosynthesis system presents an opportunity for generating minicell-coated nanoparticles with enhanced biocompatibility for diverse applications.

Total Effective Xenoestrogen Burden in Serum Samples and Risk of Endometrial Cancer in the Spanish Screenwide Case-Control Study.

BACKGROUND: Endometrial cancer is a hormone-dependent cancer, and estrogens play a relevant role in its etiology. However, little is known about the effects of environmental pollutants that act as xenoestrogens or that influence estrogenic activity through different pathways. OBJECTIVE: We aimed to assess the relationship between the combined estrogenic activity of mixtures of xenoestrogens present in serum samples and the risk of endometrial cancer in the Screenwide case-control study. METHODS: The total effective xenoestrogen burden (TEXB) attributable to organohalogenated compounds (TEXB-α) and to endogenous hormones and more polar xenoestrogens (TEXB-ß) was assessed in serum from 156 patients with endometrial cancer (cases) and 150 controls by combining chemical extraction and separation by high-performance liquid chromatography with the E-SCREEN bioassay for estrogenicity. RESULTS: Median TEXB-α and TEXB-ß levels for cases (0.30 and 1.25 Eeq pM/mL, respectively) and controls (0.42 and 1.28 Eeq pM/mL, respectively) did not significantly differ (p=0.653 and 0.933, respectively). An inverted-U risk trend across serum TEXB-α and TEXB-ß levels was observed in multivariate adjusted models: Positive associations were observed for the second category of exposure in comparison to the lowest category of exposure [odds ratio (OR)=2.11 (95% CI: 1.13, 3.94) for TEXB-α, and OR=3.32 (95% CI: 1.62, 6.81) for TEXB-ß], whereas no significant associations were observed between the third category of exposure and the first [OR=1.22 (95% CI: 0.64, 2.31) for TEXB-α, and OR=1.58 (95% CI: 0.75, 3.33) for TEXB-ß]. In mutually adjusted models for TEXB-α and TEXB-ß levels, the association of TEXB-α with endometrial cancer risk was attenuated [OR=1.45 (95% CI: 0.61, 3.47) for the second category of exposure], as well as estimates for TEXB-ß (OR=2.68; 95% CI: 1.03, 6.99). Most of the individual halogenated contaminants showed no associations with both TEXB and endometrial cancer. CONCLUSIONS: We evaluated serum total xenoestrogen burden in relation to endometrial cancer risk and found an inverted-U risk trend across increasing categories of exposure. The use of in vitro bioassays with human samples may lead to a paradigm shift in the way we understand the negative impact of chemical mixtures on human health effects. These results are relevant from a public health perspective and for decision-makers in charge of controlling the production and distribution of chemicals with xenoestrogenic activity. https://doi.org/10.1289/EHP13202.

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.

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.

Exposure to non-persistent pesticides and sexual maturation of Spanish adolescent males.

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

Cross-sectional associations of persistent organic pollutants measured in adipose tissue and metabolic syndrome in clinically diagnosed middle-aged adults.

INTRODUCTION: Although often overlooked in clinical settings, accumulation of persistent organic pollutants (POPs) in visceral adipose tissue (VAT) is thought to be a relevant risk factor for metabolic syndrome (MetS). METHODS: One hundred and seventeen patients undergoing non-oncological surgery were randomly recruited and classified as MetS + if presented 3 out of the 5 MetS components: waist circumference (WC), systolic and diastolic blood pressure (SBP and DBP, respectively), serum glucose, insulin, triglycerides (TG) and high-density lipoprotein (HDL) cholesterol levels, according International Diabetes Federation (IDF) criteria. Seventeen organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) were measured in adipose tissue samples. Linear, logistic and weighted quantile sum (WQS) regression models, adjusted for age and sex, were performed. RESULTS: One third of the participants were males (36.8%) with a median age of 44 years, showing clinical evidences of MetS (35.0%). Adjusted linear regression models showed that WC correlated positively with all OCP concentrations. Higher fasting serum glucose levels were related to higher HCB and γ-HCH concentrations. The remaining OCPs and PCBs were not associated with this MetS component. HCB was inversely associated with HDL cholesterol levels, while PCB-180 was positively associated. HCB and γ-HCH concentrations were also positively correlated with DBP and SBP levels. PCB-138 was also positively associated with SBP. Adjusted logistic models revealed that exposure to HCB and γ-HCH were associated with increased odds of MetS [ORs (95%CI) 1.53 (1.22-1.92) and 1.39 (1.10-1.76) respectively; p < 0.01]. No associations were observed for the remaining POPs. WQS models showed a positive and significant mixture effect of POPs on the odds of MetS (exp [beta] = 2.34; p < 0.001), with γ-HCH (52.9%), o,p'-DDT (26.9%) and HCB (19.7%) driving the association. CONCLUSIONS: Our findings support that POPs accumulated in VAT, specifically HCB and (gamma)-HCH, are associated with both isolated components and clinically diagnosed SMT.

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.

Childhood exposure to non-persistent pesticides and pubertal development in Spanish girls and boys: Evidence from the INMA (Environment and Childhood) cohort.

This study assessed cross-sectional associations between urinary metabolites of non-persistent pesticides and pubertal development in boys and girls from urban and rural areas in Spain and examined effect modification by body mass index (BMI). Four metabolites of insecticides (TCPy, metabolite of chlorpyrifos; IMPy, metabolite of diazinon; DETP, non-specific metabolite of organophosphates; 3-PBA, metabolite of pyrethroids) and the metabolite of ethylene-bis-dithiocarbamate fungicides (ETU) were quantified in urine collected in 2010-2016 from 7 to 11-year-old children (606 girls, 933 boys) participating in the INMA Project. Pubertal development was ascertained by Tanner stages and/or parent-reported Pubertal Development Scale (PDS). Associations between pesticide metabolites and odds of being in stage 2+ for breast development (girls), genital development (boys), pubic hair growth (girls and boys), and/or overall puberty onset, gonadarche, and adrenarche (PDS for girls and boys) were examined by mixed-effect logistic regression. Effect modification by BMI was explored by interaction terms and stratified analysis. In girls, DETP and ETU concentrations>75th percentile (P75) were associated with higher odds of overall puberty development (OR [95%CI] = 1.86 [1.07-3.24] and 1.71 [1.03-2.83], respectively, for > P75 vs. undetected concentrations), while ETU > P75 was also associated with higher odds of breast development (OR [95%CI] = 5.55 [2.83-12.91]), particularly in girls with underweight/normal weight (OR [95%CI] = 10.08 [2.62-38.76]). In boys, detection of TCPy (40%) and 3-PBA (34%) was associated with higher odds of genital development (OR [95%CI] = 1.97 [1.08-3.57] and 2.08 [1.15-3.81], respectively), and the association with 3-PBA was observed in boys with overweight/obesity alone. In addition, ETU > P75 was associated with higher odds of genital development in boys with underweight/normal weight (OR [95%CI] = 2.89 [1.08-7.74]) but higher DETP with lower odds of puberty in boys with overweight/obesity (OR [95%CI] = 0.94 [0.89-0.99] per log-unit increase in concentration). Results suggest an association of childhood exposure to ETU and certain insecticides with earlier puberty in girls and boys that may be modified by child BMI.

Concentrations and predictors of aluminum, antimony, and lithium in breast milk: A repeated-measures study of donors.

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

The Use of Human Biomonitoring to Assess Occupational Exposure to PAHs in Europe: A Comprehensive Review.

Polycyclic aromatic hydrocarbons (PAHs) are among the chemicals with proven impact on workers' health. The use of human biomonitoring (HBM) to assess occupational exposure to PAHs has become more common in recent years, but the data generated need an overall view to make them more usable by regulators and policymakers. This comprehensive review, developed under the Human Biomonitoring for Europe (HBM4EU) Initiative, was based on the literature available from 2008-2022, aiming to present and discuss the information on occupational exposure to PAHs, in order to identify the strengths and limitations of exposure and effect biomarkers and the knowledge needs for regulation in the workplace. The most frequently used exposure biomarker is urinary 1-hydroxypyrene (1-OH-PYR), a metabolite of pyrene. As effect biomarkers, those based on the measurement of oxidative stress (urinary 8-oxo-dG adducts) and genotoxicity (blood DNA strand-breaks) are the most common. Overall, a need to advance new harmonized approaches both in data and sample collection and in the use of appropriate biomarkers in occupational studies to obtain reliable and comparable data on PAH exposure in different industrial sectors, was noted. Moreover, the use of effect biomarkers can assist to identify work environments or activities of high risk, thus enabling preventive risk mitigation and management measures.

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.

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.

Concentrations and determinants of lead, mercury, cadmium, and arsenic in pooled donor breast milk in Spain.

AIM: To measure concentrations of lead (Pb), mercury (Hg), cadmium (Cd), and arsenic (As) in longitudinally collected donor breast milk samples and to determine associated factors. METHODS: Pb, Hg, Cd, and As concentrations were measured in 242 pooled breast milk samples from 83 donors to a Human Milk Bank in Spain, in 2015-2018, determining their association with the donors' sociodemographic profile, dietary and lifestyle habits, and post-partum time, among other factors, and with the nutritional characteristics of samples. Mixed-effect linear regression was used to identify predictors of Hg and As concentrations in breast milk and mixed-effect logistic regression to identify predictors of the presence of Pb and Cd. RESULTS: As was the element most frequently detected in milk samples (97.1%), followed by Hg (81.2%), Pb (50.6%), and Cd (38.0%). Their median breast milk concentrations were 1.49 µg/L, 0.26 µg/L, 0.14 µg/L, and <0.04 µg/L, respectively. Concentrations of As were higher in breast milk from primiparous donors, while Hg was higher in donors with a greater intake of fatty fish and meat and lower in samples collected after a longer post-partum time and with higher lactose content. Detection of Pb was higher among multiparous donors, those gaining weight since before pregnancy, and ex-smokers and was lower in samples collected more recently and from donors with greater intake of red meat and eggs. Cd detection was higher for donors with university education and those with greater intake of fried and canned food and more frequent use of hand cream and was lower for donors with greater bread intake. CONCLUSIONS: These findings reveal relatively high As concentrations, moderate Hg concentrations, and low Pb and Cd concentrations in pooled donor breast milk. Several factors including post-partum time, parity, smoking habit, and the intake of certain food items were associated with the metal content of milk samples.

Bisphenol-A exposure and risk of breast and prostate cancer in the Spanish European Prospective Investigation into Cancer and Nutrition study.

BACKGROUND: Bisphenol A (BPA) is an endocrine disruptor that it is present in numerous products of daily use. The aim of this study was to assess the potential association of serum BPA concentrations and the risk of incident breast and prostate cancer in a sub-cohort of the Spanish European Prospective Investigation into Cancer and Nutrition (EPIC). METHODS: We designed a case-cohort study within the EPIC-Spain cohort. Study population consisted on 4812 participants from 4 EPIC-Spain centers (547 breast cancer cases, 575 prostate cancer cases and 3690 sub-cohort participants). BPA exposure was assessed by means of chemical analyses of serum samples collected at recruitment. Borgan II weighted Cox regression was used to estimate hazard ratios. RESULTS: Median follow-up time in our study was 16.9 years. BPA geometric mean serum values of cases and sub-cohort were 1.12 ng/ml vs 1.10 ng/ml respectively for breast cancer and 1.33 ng/ml vs 1.29 ng/ml respectively for prostate cancer. When categorizing BPA into tertiles, a 40% increase in risk of prostate cancer for tertile 1 (p = 0.022), 37% increase for tertile 2 (p = 0.034) and 31% increase for tertile 3 (p = 0.072) was observed with respect to values bellow the limit of detection. No significant association was observed between BPA levels and breast cancer risk. CONCLUSIONS: We found a similar percentage of detection of BPA among cases and sub-cohort from our population, and no association with breast cancer risk was observed. However, we found a higher risk of prostate cancer for the increase in serum BPA levels. Further investigation is needed to understand the influence of BPA in prostate cancer risk.

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.

Urinary metabolites of non-persistent pesticides and serum hormones in Spanish adolescent males.

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

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.