Sex and Gender Differences on the Impact of Metabolism-Disrupting Chemicals on Obesity: A Systematic Review.

Obesity represents an important public health concern, being one of the leading causes of death worldwide. It is a multifactorial disease with many underlying intertwined causes, including genetic, environmental and behavioral factors. Notably, metabolism-disrupting chemicals (MDCs) can alter the set point control of metabolism, affecting the development and function of the adipose tissue. Epidemiological studies have reported associations between human exposure to MDCs and several altered metabolic endpoints. It is also noteworthy that sex and gender represent important risk factors in the development of obesity. Different sex-related biological and physiological characteristics influence individual susceptibility, whereas gender represents a critical component in determining the different exposure scenarios. Although some advancements in the treatment of obesity have been achieved in preclinical and clinical studies, the obesity pandemic continues to increase worldwide. The present study performed a systematic review of recent studies considering the effects of MDCs on obesity, with a specific focus on sex- and gender-related responses. This review highlighted that MDCs could differently affect men and women at different stages of life even though the number of studies evaluating the association between obesity and MDC exposure in relation to sex and gender is still limited. This evidence should urge researchers to carry out studies considering sex and gender differences. This is essential for developing sex-/gender-tailored prevention strategies to improve public health policies and reduce exposure.

Urinary Bisphenol-A (BPA) and Bis(2-ethylhexyl)phthalate (DEHP) metabolite concentrations in children with obesity: A case-control study.

Introduction Obesity is a worldwide public health problem. Experimental animal and in vitro studies suggest that the exposure to BPA and phthalates are associated to a higher risk of obesity. Objective To determine urinary excretion of bisphenol A and phthalates in obese and normal weight children. Methods A case-control study was conducted in 122 children. Sixty-six obese children 36 girls (mean age 8.41±1.27 years) and 30 boys (mean age 8.51 ± 1.33 years), and 56 normal weight children, 27 girls (mean age 7.64 ± 1.49 years) and 29 boys (mean age 7.77 ± 1.56 years) were studied. Urinary BPA and Bis(2-ethylhexyl) phthalate (DEHP) metabolites (MEHP, MEHHP and MEOHP) were measured respectively by gas chromatography and high-performance liquid chromatography. Individual determinants of exposure were evaluated through "ad hoc" questionnaires. Results BPA and DEHP metabolites were detectable in obese and normal weight children. Obese girls showed significantly higher BPA concentrations in comparison with normal weight girls (means 10.77, 95% CI 7.02-16.53 vs 5.50, 95% CI 3.93-7.71 µg/g creatinine, respectively, p< 0.02). The first step of DEHP metabolic rate was significantly higher in obese girls compared with controls (p<0.05). DEHP metabolites correlated significantly with leptin concentrations in obese girls (p< 0.03). A higher risk of obesity was found in children with BPA levels above the median values with the habit to eat food packaged (OR=11.09, 95% CI=1.28-95.78). Conclusions These findings show that a higher exposure to BPA is associated with the risk of obesity in girls. Further studies are needed to unveil the cause-effect relationship.

Special Issue "Molecular Mechanisms of Bisphenol A Toxicity and Effects of Environmental Levels on Health".

Bisphenol A (BPA) is a plasticizer that is widely used in the manufacturing of polycarbonate plastics (PC) and epoxy resins for use in a broad range of consumer products, including materials in contact with food and beverages, as well as medical devices, toys and dental sealants [...].

Exposure to Endocrine Disruptors (Di(2-Ethylhexyl)phthalate (DEHP) and Bisphenol A (BPA)) in Women from Different Residing Areas in Italy: Data from the LIFE PERSUADED Project.

Phthalates and bisphenol A (BPA) are plasticizers used in many industrial products that can act as endocrine disruptors and lead to metabolic diseases. During the LIFE PERSUADED project, we measured the urinary concentrations of BPA and Di(2-ethylhexyl)phthalate (DEHP) metabolites in 900 Italian women representative of the Italian female adult population (living in the north, centre, and south of Italy in both rural and urban areas). The whole cohort was exposed to DEHP and BPA with measurable levels above limit of detection in more than 99% and 95% of the samples, respectively. The exposure patterns differed for the two chemicals in the three macro-areas with the highest urinary levels for DEHP in south compared to central and northern Italy and for BPA in northern compared to central and southern Italy. BPA levels were higher in women living in urban areas, whereas no difference between areas was observed for DEHP. The estimated daily intake of BPA was 0.11 µg/kg per day, about 36-fold below the current temporary tolerable daily intake of 4 µg/kg per day established by the EFSA in 2015. The analysis of cumulative exposure showed a positive correlation between DEHP and BPA. Further, the reduction of exposure to DEHP and BPA, through specific legislative measures, is necessary to limit the harmfulness of these substances.

BDE-47, -99, -209 and Their Ternary Mixture Disrupt Glucose and Lipid Metabolism of Hepg2 Cells at Dietary Relevant Concentrations: Mechanistic Insight through Integrated Transcriptomics and Proteomics Analysis.

Polybrominated diphenyl ethers (PBDEs) are persistent organic chemicals implied as flame retardants. Humans are mainly exposed to BDE-47, -99, and -209 congeners by diet. PBDEs are metabolic disruptors with the liver as the main target organ. To investigate their mode of action at a human-relevant concentration, we exposed HepG2 cells to these congeners and their mixture at 1 nM, analyzing their transcriptomic and proteomic profiles. KEGG pathways and GSEA Hallmarks enrichment analyses evidenced that BDE-47 disrupted the glucose metabolism and hypoxia pathway; all the congeners and the MIX affected lipid metabolism and signaling Hallmarks regulating metabolism as mTORC1 and PI3K/AKT/MTOR. These results were confirmed by glucose secretion depletion and increased lipid accumulation, especially in BDE-47 and -209 treated cells. These congeners also affected the EGFR/MAPK signaling; further, BDE-47 enriched the estrogen pathway. Interestingly, BDE-209 and the MIX increased ERα gene expression, whereas all the congeners and the MIX induced ERß and PPARα. We also found that PBDEs modulated several lncRNAs and that HNRNAP1 represented a central hub in all the four interaction networks. Overall, the PBDEs investigated affected glucose and lipid metabolism with different underlying modes of action, as highlighted by the integrated omics analysis, at a dietary relevant concentration. These results may support the mechanism-based risk assessment of these compounds in relation to liver metabolism disruption.

Reactive Oxygen Species in the Adverse Outcome Pathway Framework: Toward Creation of Harmonized Consensus Key Events.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are formed as a result of natural cellular processes, intracellular signaling, or as adverse responses associated with diseases or exposure to oxidizing chemical and non-chemical stressors. The action of ROS and RNS, collectively referred to as reactive oxygen and nitrogen species (RONS), has recently become highly relevant in a number of adverse outcome pathways (AOPs) that capture, organize, evaluate and portray causal relationships pertinent to adversity or disease progression. RONS can potentially act as a key event (KE) in the cascade of responses leading to an adverse outcome (AO) within such AOPs, but are also known to modulate responses of events along the AOP continuum without being an AOP event itself. A substantial discussion has therefore been undertaken in a series of workshops named "Mystery or ROS" to elucidate the role of RONS in disease and adverse effects associated with exposure to stressors such as nanoparticles, chemical, and ionizing and non-ionizing radiation. This review introduces the background for RONS production, reflects on the direct and indirect effects of RONS, addresses the diversity of terminology used in different fields of research, and provides guidance for developing a harmonized approach for defining a common event terminology within the AOP developer community.

In Vitro Assessment and Toxicological Prioritization of Pesticide Mixtures at Concentrations Derived from Real Exposure in Occupational Scenarios.

Humans are daily exposed to multiple residues of pesticides with agricultural workers representing a subpopulation at higher risk. In this context, the cumulative risk assessment of pesticide mixtures is an urgent issue. The present study evaluated, as a case study, the toxicological profiles of thirteen pesticide mixtures used for grapevine protection, including ten active compounds (sulfur, potassium phosphonate, metrafenone, zoxamide, cyflufenamid, quinoxyfen, mancozeb, folpet, penconazole and dimethomorph), at concentrations used on field. A battery of in vitro tests for cell viability and oxidative stress endpoints (cytotoxicity, apoptosis, necrosis, ROS production, mitochondrial membrane potential, gene expression of markers for apoptosis and oxidative stress) was performed on two cellular models representative of main target organs of workers' and population exposure: pulmonary A549 and hepatic HepG2 cell lines. All the endpoints provided evidence for effects also at the lower concentrations used. The overall data were integrated into the ToxPI tool obtaining a toxicity ranking of the mixtures, allowing to prioritize effects also among similarly composed blends. The clustering of the toxicological profiles further provided evidence of common and different modes of action of the mixtures. The approach demonstrated to be suitable for the purpose and it could be applied also in other contexts.

Comparison of the Toxicological Effects of Pesticides in Non-Tumorigenic MCF-12A and Tumorigenic MCF-7 Human Breast Cells.

Humans are exposed to residues of organophosphate and neonicotinoid pesticides, commonly used in agriculture. Children are particularly vulnerable and, among possible adverse outcomes, the increased incidence of premature mammary gland development (thelarche) has raised concern. We evaluated the toxicological effects of chlorpyrifos (CPF), imidacloprid (IMI) and glyphosate (GLY) at exposure concentrations occurring in children on the tumorigenic MCF-7 and non-tumorigenic MCF-12A breast cell lines, as representative of the target organ model, assessing cytotoxicity, apoptosis, necrosis, intracellular reactive oxygen species (ROS) and ATP levels, 17ß-estradiol secretion and gene expression of nuclear receptors involved in mammary gland development. The pesticides decreased cell vitality in MCF-7 and cell proliferation in MCF-12A cells. ATP levels were decreased in MCF-7 cells by pesticides and apoptosis was increased in MCF-12A cells only by GLY (2.3 nM). ROS production was decreased by pesticides in both cell lines, except IMI (1.6 nM) in MCF-7 cells. Endocrine disrupting activity was highlighted by induction of 17ß-estradiol secretion and modulation of the gene expression of estrogen alpha and beta, progesterone, androgen, and aryl hydrocarbon receptors in both cell lines. The use of MCF-7 and MCF-12A cells highlighted dissimilar modes of action of each pesticide at low human relevant concentrations.

Italian Children Exposure to Bisphenol A: Biomonitoring Data from the LIFE PERSUADED Project.

A human biomonitoring (HBM) study on bisphenol A (BPA) in Italian children and adolescents was performed within the LIFE PERSUADED project, considering the residing areas, sex and age. The median urinary BPA level was 7.02 µg/L, with children living in the South of Italy or in urban areas having higher levels than those residing in the North or in rural areas. Children aged 4-6 years had higher BPA levels than those aged 7-10 and 11-14 years, but no differences were detected between sexes. The exposure in Italian children was higher compared to children from other countries, but lower than the HBM guidance value (135 µg/L). The estimated daily intake was 0.17 µg/kg body weight (bw) per day, about 24-fold below the temporary Tolerable Daily Intake of 4 µg/kg bw per day established by the European Food Safety Authority. However, this threshold was exceeded in 1.44% of the enrolled children, raising concern about the overall exposure of Italian young population.

Lifestyle Habits and Exposure to BPA and Phthalates in Women of Childbearing Age from Northern Italy: A Pilot Study.

Background: Endocrine-disrupting chemicals (EDCs) are compounds that interfere with aspects of hormonal signaling. Considerable attention has been paid to their biological effects especially in women of childbearing age or during pregnancy as EDCs have been reported to cross the placenta becoming concentrated in the fetus' circulation. Lifestyle habits, daily consumption of packaged foods and use of healthcare/cosmetic products are associated with increased EDCs levels. This cross-sectional research examined the EDCs levels and the lifestyle determinants of EDC exposure in a cohort of reproductive-age women from Northern Italy. Methods: Forty-five women (median age: 36, IQR: 30-38) were evaluated for urinary bisphenol A (BPA) and phthalates levels and also studied for EDCs' major determinants of daily exposure; food frequency/dietary, physical activity, smoking habits and weight status. Results: Although 100% of women seemed to have been exposed to common sources of EDCs, they reported a healthy lifestyle. The multivariable model described a positive and significant association between consumption of sauces/dressings in plastic containers and monoethyl phthalate exposure (p = 0.037). Conclusions: Since reproductive age encompasses a critical window for future health and functioning of the "mothers-to-be" and their children, future studies on prenatal dietary BPA and phthalate exposure and the role of consumer product choices in reducing such exposure are recommended.

Toxicological Assessment of Oral Co-Exposure to Bisphenol A (BPA) and Bis(2-ethylhexyl) Phthalate (DEHP) in Juvenile Rats at Environmentally Relevant Dose Levels: Evaluation of the Synergic, Additive or Antagonistic Effects.

BACKGROUND: The general population (including children) is exposed to chemical mixtures. Plasticizers such as Bisphenol A (BPA) and Phthalates (mainly Bis(2-ethylhexyl) phthalate-DEHP) are widespread contaminants classified as endocrine disrupters which share some toxicological profiles and coexist in food and environment. METHODS: To identify hazards of DEHP and BPA mixtures, the juvenile toxicity test-where rodents are in peripubertal phase of development, resembling childhood-was selected using exposure data from biomonitoring study in children. Biological activity and potential enhanced and/or reduced toxicological effects of mixtures due to common mechanisms were studied, considering endpoints of metabolic, endocrine and reproductive systems. The degree of synergy or antagonism was evaluated by synergy score calculation, using present data and results from the single compound individually administered. RESULTS: In metabolic system, synergic interaction predominates in female and additive in male rats; in the reproductive and endocrine systems, the co-exposure of BPA and DEHP showed interactions mainly of antagonism type. CONCLUSIONS: The present approach allows to evaluate, for all the endpoints considered, the type of interaction between contaminants relevant for human health. Although the mode of action and biological activities of the mixtures are not completely addressed, it can be of paramount usefulness to support a more reliable risk assessment.

Metabolic, reproductive and thyroid effects of bis(2-ethylhexyl) phthalate (DEHP) orally administered to male and female juvenile rats at dose levels derived from children biomonitoring study.

Bis(2-ethylhexyl) phthalate (DEHP) is a plasticizer used in several items, non-covalently bound to plastics and easily released, since metabolites were found in human matrices. DEHP is an endocrine disrupter and children are particularly vulnerable and susceptible to DEHP effects due to higher exposure levels and developmental stage. A juvenile toxicity study was performed to identify DEHP hazard and mode of action in Sprague-Dawley rats of both sexes during peri-pubertal period - corresponding to childhood phase - from weaning, post-natal day (PND) 23, to full sexual maturity (PND60); the dose levels of 0, 9, 21 and 48 mg/kg bw/day were derived from LIFE PERSUADED biomonitoring study in children. DEHP was administered by gavage for 28 days (5 days/week); timing of preputial separation and vaginal opening was observed during treatment. Histopathological analysis was performed on: adrenals, spleen, liver, thyroid and reproductive organs. The following serum biomarkers were assessed: estradiol, testosterone, anti-Mullerian hormone, tetraiodothyronine, thyroid stimulating hormone, adiponectin and leptin. Gene expression on hypothalamic-pituitary area was focused on follicle stimulating, luteinizing, and thyroid stimulating hormones. The results showed that main targets of DEHP during juvenile period were liver and metabolic system in both sexes, while sex-specific effects were recorded in reproductive system (male rats) and in thyroid (female rats). DEHP exposure during peri-pubertal period at dose levels derived from biomonitoring study in children can induce sex-specific imbalances identifying the juvenile animal model as a sound tool to identify hazards for a reliable risk assessment targeted to children.

Integrated Approach to Evaluate the Association between Exposure to Pesticides and Idiopathic Premature Thelarche in Girls: The PEACH Project.

Several pesticides are recognized as endocrine-disrupting chemicals (EDCs) since they can interfere with the dysregulation of sexual, thyroid and neuro-endocrine hormones. Children are particularly vulnerable to the adverse effects of EDCs due to their developmental stage, peculiar lifestyle and dietary habits. In this context, the exposure to pesticides represents an important risk factor associated with early development. This study deals with the possible association between exposure to pesticides and idiopathic premature thelarche in girls from areas of intensive agriculture practice in the Centre of Italy. An integrated approach was set up, including: (i) a case-control study on girls with idiopathic premature thelarche; (ii) the evaluation of multiple pesticides exposure in girls; (iii) the evaluation of multiple pesticides in food; (iv) the dietary intake of pesticide residues; (v) the assessment of toxicological effects of widely used pesticides by in vitro model. Data integration will provide an estimate of the predictive risk of potential effects on girls' health, linked to dietary intake.

Biomonitoring of Bis(2-ethylhexyl)phthalate (DEHP) in Italian children and adolescents: Data from LIFE PERSUADED project.

The Bis(2-ethylhexyl)phthalate (DEHP), a widespread plasticizer, is considered an endocrine disrupting chemical with main toxicological effects on reproductive and metabolic systems. Human biomonitoring (HBM) studies are promoted to evaluate the background exposure levels. In the frame of LIFE PERSUADED project, the HBM study measured DEHP main metabolites (mono-(2-ethylhexyl) phthalate, MEHP; 2-ethyl-5-hydroxy-hexylphthalate, MEHHP; 2-ethyl-5-oxo-hexylphthalate, MEOHP) in Italian children and adolescent (4-14 years old) according to geographical macro-areas and areas, age and sex. Children from the South and the Centre of Italy showed higher median levels of DEHP, as a sum of its metabolites (48.14 and 47.80 µg/L), than those from the North (39.47 µg/L; p = 0.0090 and 0.0004, respectively). Considering the total population, boys are more exposed than girls (only as urinary volume), and children aged 4-6 years have higher median levels than those 7-10 and 11-14 years old. The derived reference values (RV95) for DEHP in children is 168 µg/L. The relative metabolic rates of DEHP, the background levels and, thus, the RV95, vary with the geographical area, age and sex, indicating that all these parameters should be considered in the risk assessment.

Juvenile Toxicity Rodent Model to Study Toxicological Effects of Bisphenol A (BPA) at Dose Levels Derived From Italian Children Biomonitoring Study.

Bisphenol A (BPA) is a plasticizer with endocrine disrupting properties particularly relevant for children health. Recently BPA has been associated with metabolic dysfunctions but no data are yet available in specific, long-term studies. This study aimed to evaluate BPA modes of action and hazards during animal juvenile life-stage, corresponding to childhood. Immature Sprague-Dawley rats of both sexes were orally treated with 0 (vehicle only-olive oil), 2, 6, and 18 mg/kg bw per day of BPA for 28 days, from weaning to sexual maturity. Dose levels were obtained from the PERSUADED biomonitoring study in Italian children. Both no-observed-adverse-effect-level (NOAEL)/low-observed-adverse-effect-level (LOAEL) and estimated benchmark dose (BMD) approaches were applied. General toxicity, parameters of sexual development, endocrine/reproductive/functional liver and kidney biomarkers, histopathology of target tissues, and gene expression in hypothalamic-pituitary area and liver were studied. No mortality or general toxicity occurred. Sex-specific alterations were observed in liver, thyroid, spleen, leptin/adiponectin serum levels, and hypothalamic-pituitary gene expression. Thyroid homeostasis and liver were the most sensitive targets of BPA exposure in the peripubertal phase. The proposed LOAEL was 2 mg/kg bw, considering as critical effect the liver endpoints, kidney weight in male and adrenal histomorphometrical alterations and osteopontin upregulation in female rats. The BMD lower bounds were 0.05 and 1.33 mg/kg bw in males and females, considering liver and thyroid biomarkers, respectively. Overall, BPA evaluation at dose levels derived from children biomonitoring study allowed to identify sex-specific, targeted toxicological effects that may have significant impact on risk assessment for children.

The LIFE PERSUADED project approach on phthalates and bisphenol A biomonitoring in Italian mother-child pairs linking exposure and juvenile diseases.

Phthalates and bisphenol A (BPA), plasticizers used in several products of daily life, are considered as endocrine disrupters, therefore children exposure is particularly relevant. The LIFE PERSUADED project aims to define the following: (a) the evaluation of internal levels of DEHP's metabolites and BPA in Italian children and their mothers, (b) the association of the exposure with puberty development and obesity diseases, and (c) the effects of exposure in juvenile in vivo model. The cross-sectional study has involved 2160 mother-child pairs, including males and females, children and adolescents, from urban and rural areas of North, Center, and South Italy. A structured questionnaire and a food diary are designed to evaluate the association between lifestyle variables potentially related to DEHP/BPA exposure and internal levels, through univariate and multivariate analyses. Two pilot case-control studies are carried out on idiopathic premature thelarche and precocious puberty (30 girls each group, aged 2-7 years) and idiopathic obesity (30 boys and 30 girls, aged 6-10 years), matched to healthy controls. BPA and DEHP's metabolites are analyzed in urine samples from all recruited subjects. Clinical and toxicological biomarkers are evaluated in serum of case-control subjects. Moreover, the toxicity study is carried out in a juvenile rodent model exposed to mixtures of BPA and DEHP at dose levels recorded in children population. The scientific results of LIFE PERSUADED will contribute to risk assessment of BPA and DEHP.

[A network of LIFE projects to promote the transfer and exchange of knowledge on environment and health]. / Un network di progetti LIFE per promuovere il trasferimento e lo scambio di conoscenze su ambiente e salute.

Eleven projects within the LIFE programme (through which the Directorate-General for Environment of the European Commission provides funding for projects aim at protecting environment and nature) addressing environmental-health-related issues have been involved in a collaborative network called KTE LIFE EnvHealth Network. The shared issues tackled by that projects are knowledge transfer and exchange (KTE). The objective of the LIFE programme is to support the implementation of the environmental legislation in the European Union, to provide new tools and knowledge that will help to better protect both the territory and the communities. Transferring knowledge to decision makers, at the appropriate and effective level, is therefore a central function of the projects. The Network promotes national and international networking, which intends to involve other projects, to provide methodological support, to make information and successful practices circulate, with the aim of multiplying the energies of each project involved.

Biomonitoring of chemicals in biota of two wetland protected areas exposed to different levels of environmental impact: results of the "PREVIENI" project.

The PREVIENI project (funded by the Ministry of Environment) investigated the exposure to endocrine disrupters in samples of human population and environmental biota in Italy. The environmental biomonitoring considered two Italian WWF Oasis, with the aim to compare the presence and effects of endocrine disruptors in organisms from two protected natural areas, respectively, upstream and downstream a chemical emission site. Chemical analysis of pollutants' tissue levels was made on tissues from earthworm, barbell, trout, and coot, selected as bioindicator organisms. The contaminants considered were as follows: the perfluorinated compounds perfuoroctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), polychlorinated biphenyls (PCBs 58 congeners), polybrominated diphenyl ethers (PBDEs, 13 congeners), polycyclic aromatic hydrocarbons (PAHs, 16 compounds), toxic trace elements, the phthalate di-2-ethylexyl phthalate (DEHP) and its primary metabolite, bisphenol A, synthetic musk compounds (musk xylene, musk ketone, tonalide, and galaxolide), and p-nonylphenol. The analyses showed low concentrations of most pollutants in all species from both areas, compared to available literature; noticeable exceptions were the increases of DEHP's primary metabolite, PBDE, PAHs, Hg, and Pb in barbells, and of PCB and Cd in earthworms from the downstream area. The results showed the presence of endocrine disruptors, including those considered as "non-persistent," in bioindicators from protected areas, albeit at low levels. The results provide a contribution to the evaluation of reference values in biota from Mediterranean Europe and support the relevance of monitoring exposure to pollutants, in particular for freshwater environment, also in protected areas.