Hypiend - Multicomponent Behavioral Intervention in Pre-puberal Children (Hypiend-PPC)

• BACKGROUND AND JUSTIFICATION

1.1. SCOPE AND ASSESSMENT OF CURRENT KNOWLEDGE DIRECTLY RELATED TO THE SCIENTIFIC QUESTIONS TO BE ANSWERED IN THE CLINICAL STUDY

Several epidemiological studies have identified concerning links between high endocrine-disrupting chemicals (EDCs) exposure and adverse health effects in children and adolescents. As an illustrative example, in the framework of the Human Biomonitoring for Europe (HBM4EU) project, Lange et al. (2022) analysed the risk assessment of combined exposure to anti-androgen phthalates (Di(2-ethylhexyl) phthalate (DEHP), Di-isobutyl phthalate (DiBP), Di-n-butyl phthalate (DnBP), Butyl benzyl phthalate (BBzP) and Di-isononyl phthalate (DiNP) in children (6-11 years) and adolescents (12-18 years) from various European countries. The authors reported that DnBP and DiBP were the main drivers of the increased hazard index (HI) for a phtalate mixture in a large cohort of European children and adolescents. Notably, for 63% of these subjects with HI > 1, this risk would not have been detected if chemicals had been analyzed individually, illustrating the importance of mixture risk assessments. In addition, the authors also found regional differences in exposure-related HI, with children and adolescents in eastern Europe more exposed than those in the south and west, highlighting the need to include countries with varying exposure levels for more comprehensive conclusions and tailored measures, policies and recommendations.

Various epidemiological studies suggest that increased exposure to EDCs with hormonal activity in recent decades, especially to substances with direct estrogenic activity, may contribute to the observed earlier onset of puberty in girls and boys in developed countries. In this regard, a very recent cross-sectional study of 1539 Spanish children found that higher concentrations of certain non-persistent pesticides were associated with greater probability of developing earlier puberty in both boys and girls, with this association modulated by the child's body mass index (BMI).

To the investigators' knowledge, data on interventions aimed at reducing EDC exposure in prepubertal children are scarce and inconclusive, including most of them under 100 children, with most studies including fewer than 100 children, analyzing at most two EDC families, and lacking control groups.

HYPIEND aims to evaluate whether a multicenter, multicomponent controlled intervention (consisting of the implementation of plastic-free lunchrooms in elementary schools, school-based educational programs for children, parent workshops, and a mobile application for parents to limit EDC exposure) conducted in primary schools with prepubertal children, their parents and the school staffs can reduce urinary concentrations of various EDCs (focusing first on bisphenol A as the primary outcome) and potnetially prevent earlier puberty onset associated with high EDC exposure. In addition, various anthropometric measurements will also be assessed. Effects will be compared to a control group receiving only placebo workshops on screen dangers, physical activity benefits, and good sleep habits. The intervention will be implemented over two and a half consecutive school years (from November 2025 (start of the intervention) to December 2027) and include behavior change techniques and models to achieve high adherence to the intervention.

Schools will be recruited from rural and urban environmental zones to account for varying contaminant exposures. Investigators will measure urinary biomarkers of exposure to different EDC families at three time points (baseline and end of the first and third school years). A risk assessment of EDC exposure will be performed, including all data obtained in urine from these time points.Multivariate analyses will explore predictive variables for EDC exposure, such as dietary and behavioral habits, school environment, and socioeconomic status. The study will be conducted in two countries (Belgium and Spain). Overall, this approach will contribute to obtaining a more accurate picture of EDC exposure and the degree of EDC exposure reduction in response to the implemented intervention, considering key contributing factors in EDC-related burden.

1.2. RESULTS OF COMPLETED CLINICAL STUDIES AND NUMBER OF ONGOING CLINICAL STUDIES USING THE SAME INTERVENTION IN THE SAME INDICATION

Recently, Sessa et al. (2021) demonstrated that a school lunchroom intervention (implementation of a plastic-free service) for 5 days per week over 6 months significantly decreased urinary bisphenol A levels both between groups and within the group enrolled in the plastic-free school lunchroom. However, larger MBI studies involving schools and families, supported by experts in behavior change and educational sciences, are needed to promote long-lasting effects and minimize overall exposure to different EDCs more conclusively.

1.3. LEVEL OF EVIDENCE RELATED TO THE MECHANISM OF ACTION OF THE INTERVENTION IN THE PLANNED CLINICAL STUDY POPULATION

EDC-Mix-Risk and ENDpoiNTs are two outstanding Horizon 2020 projects that combined experimental research in both cells and animals with epidemiological data. Researchers from both projects and others demonstrated, a multivariate regression statistical model with data from the SELMA pregnancy study, that different EDCs (including phthalates, alkylphenols and perfluoroalkyl substances) analyzed in the urine or serum of women at week 10 of pregnancy were associated with language delay in children at 2.5 years of age. Subsequently, the identified EDCs were mixed and tested in human brain organoids, as well as in Xenopus leavis and Danio rerio to elucidate the molecular and functional impact of exposure. Finally, the authors integrated experimental and epidemiological data and conducted a risk assessment, finding increased odds of language delay in up to 54% of offspring with prenatal exposures above experimentally derived levels of concern.

Despite these highly relevant findings, few human studies have shed light on the mechanisms underlying EDC-mediated dysfunctions in the HPA axis, including induction of epigenetic changes in key genes involved in neuroendocrine regulation and precocious puberty, as well as alterations in inflammatory response and gut microbiota (dysbiosis). EDCs may increase the risk of neurodevelopmental disorders in children by interfering with estrogen and thyroid hormone signaling or metabolism in early life.Very recently, increased urinary levels of a mixture of environmental chemicals (phenols, toxic metals, and non-persistent pesticides) measured in adolescents aged 15-17 years were associated with higher circulating levels of kisspeptin-54 protein and lower methylation levels at different cytosine-phosphate-guanine (CpG) sites in the kiss1 gene. In addition, adolescents with higher serum kisspeptin-54 had elevated luteinizing hormone (LH) levels, and third-tertile CpG1, CpG2, and total CpG methylation percentages were associated with lower follicle-stimulating hormone (FSH) and estradiol (E2) levels. These findings suggest that kisspeptin protein, which is a precursor-derived neuroactive peptide (pro-hormone, pre-pro-kisspeptin) released by neurons in the hypothalamus, could be a promising biomarkers of the effects of environmental chemical mixtures on the reproductive hormone profile.

However, despite this evidence, to the knowledge of the investigators, no human intervention studies aimed at reducing EDC exposure have analyzed the molecular impacts that may accompany these changes in EDC exposure. Further research is needed to shed more light on these questions, including molecular and omics analyses in humans to determine whether human intervention studies aimed at reducing EDC exposure acan favorably modulate these molecular changes.

In this project, various molecular and omics approaches will be conducted to shed more light, in the framework of the HPA axis, on the epigenetic drivers most susceptible to alteration and the interaction between gut microbiota and the impact of EDC exposure on systemic inflammation. These analyses will be performed on samples obtained in the study, taking into account results obtained in preclinical models exposed to EDC mixtures that attempt to mimic real human exposure, to increase the chances of obtaining robust and reliable results using a targeted approach. Specifically, in prepubertal children, in addition to gut microbiota, the investigators will also assess HPA axis-related hormones, methylation of different CpG sites of key genes involved in the HPA axis and circulating kisspeptin levels using blood samples. Overall, these analyses will enable a deeper understanding of the mechanisms by which EDCs exert their harmful effects, as well as how intervention studies aimed at reducing exposure to these chemicals can modulate these biological pathways and the identified molecular biomarkers of exposure. To the knowledge of the investigators, this approach has not yet been implemented in large human intervention studies in the EDC field.

1.4. HYPIEND PROJECT

The intervention presented here is part of the European HYPIEND Project. This project is coordinated by the Technological Center of Catalonia (Eurecat), funded by the Horizon Europe framework program (Grant agreement ID: 101137440).

The HYPIEND project investigates the effects of exposure to multiple EDCs on the function and epigenetic programming of the HPA axis to delineate intervention strategies to minimize exposure and consequences on the neuroendocrine system during the perinatal and pre-pubertal stages.

The multidisciplinary HYPIEND consortium comprises 14 partners from eight European countries. Among them: Stichting Radboud Universiteit (Netherlands), Institut de Investigació en Ciencies de la Salut Germans Trias i Pujol (Spain), Sciensano (Belgium), Kungliga Tekniska Hoegskolan (Sweden), Katholieke Universiteit Leuven (Belgium), University of Granada (Spain), Centrum Medyczne Ksztalcenia Podyplomowego (Poland), Protoqsar 2000 sl (Spain), Enco srl (Italy), Fundacion para la investigacion del hospital universitario la Fe de la Comunidad Valenciana (Spain), Université de Liège (Belgium), Universite de Geneve (Switzerland), King's College London (UK). More information about this project can be found at the following links: https://hypiend.eu/

Here is an overview of the Hypiend methodology. Data from public databases will be combined to develop a knowledge base linking selected EDC exposure to health outcomes related to the HPA axis. Quantitative structure-activity relationship (QSAR) methodologies will be combined with data analysis to identify co-exposure patterns. EDC mixtures with high prevalence in the target populations will be defined, both qualitatively and quantitatively, based on these coexposure patterns. Candidate EDC mixtures will be tested in cell-based models to define dose-response patterns and dosing for subsequent studies. Zebrafish models recapitulating HPA axes will be used to select those EDC mixtures with the greatest impact. Next, selected EDC mixtures will be tested in placenta and blood-hypothalamus barrier (BHE) models to assess the diffusion of EDC mixtures and characterize the fractions reaching both the fetus and hypothalamus. Effects on hypothalamus and pituitary development and function will be assessed in stem cell-derived organoids and organoids and organs-on-chip (OoC), with particular attention to effects on whole genome DNA methylation and hormone functionality. The results will be used to further select complex mixtures of EDCs based on their HPA epigenetic programming and disruptive potential. Subsequently, laboratory animals will be exposed to EDC mixtures from gestation to first generation to assess the programming and cumulative effects of EDC exposure on the HPA axis. Physiological and behavioral outcomes of HPA disruption will be monitored. In parallel, effects on DNA methylation in hypothalamus, pituitary and peripheral blood cells, as well as changes in specific circulating ECV, will be determined. An artificial intelligence (AI)-based systems biology approach will be used to describe associations between hypothalamus/pituitary function, hematoperipheral blood cell methylation patterns, and multiomics data. These associations will be used to establish specific signatures associated with HPA disruption by EDC exposure. Signatures will be explored in samples from the target population (i.e., children in their first 1000 days and prepubertal) to assess their translatability and application as biomarkers. Human studies will not be limited to cross-sectional determinations, but are intended as novel interventions to promote decreased EDC exposure and assess cause-effect relationships. This will enable the definition of biomarkers based on data obtained from the different work packages and a systems biology approach to unravel conserved mechanisms of action.

The work has been proposed as a sequence of tasks aimed at defining the impact of realistic EDC co-exposure on the HPA axis during two target life stages, i.e. prepubertal and perinatal. The different levels proposed by organizations such as OECD, including new approach methodologies (NAMs) and computational toxicology, have been adopted to minimize animal research. This design combines translational research with intervention studies.

• HYPOTHESIS OR STUDY QUESTION

A MBI in primary schools in two European countries (Spain and Belgium) over 28 months will reduce urinary EDC levels in prepubertal children and increase knowledge about these contaminants.

• OBJECTIVES

3.1. MAIN OBJECTIVE

The overall goal of HYPIEND is to understand the effects of EDC co-exposure on HPA axis function and epigenetic programming in order to delineate intervention strategies to minimize exposure and neuroendocrine consequences during the perinatal and prepubertal stages.

In this framework, the main objective of this specific clinical study is to demonstrate that a MBI implemented in primary schools in two different European countries during 25 months is effective in reducing urinary EDC levels in prepubertal children. This will be achieved using the HAPA constructs to improve the intervention and parents' knowledge of these contaminants, as well as to advance scientific understanding.

3.2. SECONDARY OBJECTIVES

• To determine whether an intervention designed to reduce EDC exposure can prevent deleterious effects related to altered HPA function in prepubertal children aged 6 to 8 years (earlier onset of puberty and alterations in BMI and cognitive function).
• To better understand the relationship between urinary EDC levels and HPA-related hormone levels, as well as DNA methylation patterns and gut microbiota composition, thereby shedding light on the mechanisms by which EDCs exert their deleterious effects.
• To explore, through multivariate and risk assessment analyses, predictors of elevated urinary EDC levels.
• To determine whether an intervention based on the combined HAPA and PBS models in primary schools effectively improves HAPA constructs, knowledge, and behaviors of parents, children, and educational teams toward these pollutants, compared to a placebo intervention.

Inclusion criteria

• For children: aged between 6 and 8 from participating schools, and their parents/legal guardians.
• For parents/legal guardians:

• Understanding the local languages (either French in Belgium, or Spanish/Catalan in Spain) and being able to complete forms in that language;
• Be in possession of a smartphone. However, if participants do not have a smartphone and wish to do so, a smartphone will be made available to them for the duration of the study.

For schools:

• Fewer than 5 participating children enrolled in the school.
• Unwillingness or inability to devote sufficient time to the project.

For children:

• Refusal to participate (child informed consent form not obtained); parents will be asked to discuss with the child before exclusion.
• Parents or legal guardians do not sign the informed consent form.

For parents/legal guardians:

• Refusal to participate.
• Unwillingness to use a mobile phone as part of the project (exclusion from sample).