(Formerly Section for Developmental and Environmental Toxicology, Institute of Pharmacology and Toxicology, University of Zurich)
Humans and animal populations are increasingly exposed to chemicals in the environment throughout their lifetime, during reproduction and ontogeny. GREEN Tox (Group for Reproductive, Endocrine and Environmental Toxicology) conducts research at different levels: (1) longterm toxicity of environmental chemicals (including cosmetics and flame retardants) with focus on endocrine effects, the developing organism, brain and reproduction, (2) analysis of internal exposure to environmental chemicals in human populations, and (3) development of new refined test methods for the characterization of endocrine activity and developmental toxicity of chemicals.
Rational actions require reliable information. We are therefore working along two lines: conducting research and providing information to the scientific community and to the public.
(Horizon 2020, Grant No. 825759)
Web site: https://endpoints.eu/
Exposure to environmental chemicals with endocrine activity (endocrine disrupting chemiclas, EDCs) during early development has been documented to be linked with impaired development of brain and behavior in epidemiological studies on children and in experimental animal models. Yet, current hazard assessment of EDCs does not address developmental neurotoxicity (DNT). Thus, there is an urgent need for novel testing and screening tools to address endocrine disruption (ED)-induced developmental neurotoxicity, based on new scientific knowledge.
ENDpoiNTs addresses this issue. With 16 participants in Europe, USA and Australia, the project integrates expertise in endocrine disruption and developmental neurotoxicity, and combines state-of-the-art in silico and in vitro tools, animal models, and human epidemiological and biomonitoring data. The project will proceed in three phases: 1. In a first step, molecular effect patterns of endocrine active chemicals will be studied in cell culture systems with neural and glial cell precursors, and in in vivo models of developmental neurotoxicity, and will be compared with molecular markers identified in epidemiological studies, in order to establish correlative and causal links between endocrine disruption and DNT. 2. Subsequently, in silico tools and new in vitro models for chemical screening, and novel molecular endpoints for existing animal-based test guidelines, will be developed by integrating in vitro, in vivo, and human omics data with behavioral outcomes. Newly developed models will be assessed by an internal validation procedure. Human relevance will be ensured by linking experimental and epidemiological evidence. 3. In the implementation phase of the project, an integrated testing approach will be developed, based on the newly developed in silico analyses and in vitro assays, and new molecular endpoints in in vivo assays. Regulatory relevance will be ensured by developing strategies for implementation of these tools and endpoints into regulatory frameworks.
GREEN Tox has two main tasks in the project:
1. In vivo rat model
The developmental in vivo rat model has been designed by GREEN Tox and is carried out in collaboration with the Universidad Complutense in Madrid (Jesus Tresguerres). Rats are exposed in the food before mating and during gestation and lactation to low and moderate doses of environmental chemicals that have been found to be correlated with behavioral disturbances in children. The brains of male and female offspring are analyzed at the neonatal stage and in adulthood by GREEN Tox, and behavior of offspring will be studied by Madrid. Two regions will be studied in developing and adult brain, hippocampus, which plays a central role in learning and memory, and medial preoptic area, which is involved in the control of sexual behavior and gonadal function. Development of both brain areas is regulated by sex hormones and thyroid hormones. The same piece of brain tissue will be analyzed for genome-wide gene expression (transcriptomics) by GREEN Tox, for epigenetic changes by Uppsala University (Joëlle Rüegg), and for metabolomics by Stichting VU (Vrije Universiteit) Amsterdam (Pim Leonards). Non-coding RNAs will also be investigated. Epigenetic, transcriptomic, and metabolomic data will be analyzed in relation to chemical exposure and behavioral outcome in the offspring, and will further be compared with molecular data from human epidemiological studies, and with data from the in vitro models, in order to identify toxicologically relevant molecular patterns or single molecular endpoints of predictive value.
2. “Implementation: Novel strategies for regulatory testing of EDC-related DNT”.
GREEN Tox will lead the work package on implementation, which aims at developing new strategies of regulatory testing for EDC-related developmental neurotoxicity, by integrating the new models and molecular endpoints developed in the project into regulatory hazard and risk assessment.
AOPs are designed to link molecular events at the cellular level (initiating events) through intermediary steps at higher levels of organization (e.g., tissue, organ) with adverse outcomes at the organism or population level. The AOP concept aims to enhance the predictive value of in vitro tests by establishing links between processes analyzed in vitro and toxicity endpoints at the organism level. GREEN Tox has started a project, funded by the Swiss Health Office, in collaboration with the Universidad Complutense in Madrid (Depto de Fisiologia) and the Functional Genomics Center of the University of Zurich and Swiss Federal Institute of Technology Zurich (ETH) to develop an AOP linking behavioral outcomes resulting from developmental exposure to chemicals, with molecular actions analyzed in in vitro cell systems. Learning and memory represent a suitable adverse endpoint because impairments are well documented in experimental animals after developmental exposure to different classes of chemicals, and because cognitive functions are endpoints in OECD testing guidelines. The model is relevant for humans, as exposure to a number of chemicals has been correlated with analogous cognitive deficiencies in epidemiological studies in humans. Since the hippocampus plays a central role in this type of behaviors, we are investigating chemical-induced changes in gene expression and epigenetic markers of the developing hippocampus as a possible link between adverse outcome and initiating events at the cellular level. Late effects at the molecular level have been identified in adult rodent hippocampus after exposure to chemicals during early development. Development and function of the hippocampus also depend on hormone actions (estrogens, androgens, thyroid hormones), which means that a hippocampus- based AOP could be expected to also detect effects of endocrine disrupting chemicals on brain development.
During the past decade, researchers realized that chemicals out of different classes, derived from human sources (pesticides, industrial chemicals, drugs) or plants (phytoestrogens), are capable to activate or block hormone receptors, in particular estrogen or androgen receptors. Chemicals may further interfere with synthesis, transport or breakdown of hormones. Such chemicals may affect hormone-regulated developmental processes in peripheral organs (reproductive tract) and in central nervous system (e.g., sexually dimorphic neuron groups), which may result in alterations of sexual behavior or other types of behavior.
Chemical-induced developmental alterations have been observed in wildlife, and more recently, effects on humans have been demonstrated in selected cases. Risk assessment of endocrine active chemicals requires identification and characterization of endocrine activity, assessment of toxicity in particular with respect to long-term effects, and analysis of exposure levels in environmental samples and biosphere, including humans.
GREEN Tox participated in the EU 7th Framework Programme project "Contaminant mixtures and human reproductive health - novel strategies for health impact and risk assessment of endocrine disrupters" (CONTAMED) (see Links). This project addresses a major unsolved problem posed by endocrine active chemicals (endocrine disrupting chemicals, EDCs): Humans and animals are exposed to mixtures of EDCs. So far, risk assessment for registration of chemicals is based on studies conducted with single chemicals. In order to be able to assess the real health risks of EDCs, procedures for the evaluation of the combined risk of mixtures of EDCs need to be developed. CONTAMED explored the hypothesis that exposure to EDC mixtures in fetal life may lead to adverse delayed impacts on human reproductive health. To achieve this goal, CONTAMED combined epidemiological approaches with laboratory science.
The work plan for CONTAMED is organised in three major strands focusing on human studies (assessment of exposure to EDCs), animal models (effects of EDC mixtures on reproduction and development of rats, with EDC mixtures designed on the basis of human exposure studies), and in vitro assays including metabolomics.
GREEN Tox investigated the effect of three different EDC mixtures, i.e. a mixture of chemicals with anti-androgenic activity (A-Mix), a mixture of chemicals with estrogenic activity (E-Mix) and a combined mixture containing all chemicals (T-Mix) on the developing brain in a rat model, in collaboration with the Technical University of Denmark (see Christiansen et al., 2012). We analyzed gene expression in two sexually dimorphic brain regions (medial preoptic area and ventromedial hypothalamic nucleus) during early postnatal development by microarrays and real time RT PCR. The three EDC mixtures displayed gene expression patterns that were specific for the individual mixture, as well as for sex and brain region. All mixtures had a strong, mixture-specific impact on genes encoding for components of excitatory glutamatergic synapses and genes controlling migration and pathfinding of glutamatergic and GABA-ergic neurons, as well as genes linked with increased risk of autism spectrum disorders. Because development of glutamatergic synapses is regulated by sex steroids also in cortical areas (hippocampus), the developing glutamatergic synapse may represent a general target of ECD mixtures.
Reference: Lichtensteiger et al., Endocrinology 156: 1477–1493, 2015.
Many chemicals in cosmetics are high production volume chemicals. Their dual role as compounds administered to humans and as chemicals released into the environment and entering the food chain has long been neglected. We follow a triple strategy, identification of endocrine activity of individual chemicals, investigation of longterm effects with emphasis on reproduction and ontogeny as particularly sensitive life-stages, and analysis of internal human exposure (for review, see Schlumpf et al., 2008).
Endocrine activity and developmental toxicity of UV filters,
After the identification of a number of UV filters as estrogenic or androgenic in vitro and in in vivo (Schlumpf et al., 2001, Ma et al., 2003) and the characterization of two of them, 4-methylbenzylidene camphor (4-MBC) and 3-benzylidene camphor (3-BC) as preferential estrogen receptor (ER) beta ligands (Schlumpf et al., 2004), we studied developmental toxicity of 4-MBC and 3-BC in a transgeneration rat model. Pre- and postnatal exposure to 4-MBC was found, i.a., to increase prostate size in neonatal rats (Hofkamp et al., 2008), delay male puberty (Durrer et al., 2007), and affect reproductive organ weights and estrogen target gene expression in adult offspring of both sexes (Durrer et al., 2005, 2007). 3-BC also influenced male puberty, reproductive organ development and gene expression with partly different effect patterns (Schlumpf et al., 2008). Both, 4-MBC and 3-BC impaired female sexual behavior in pre- and postnatally exposed offspring (Faass et al., online in press), and gene expression in sexually dimorphic brain regions (Maerkel et al., 2005, 2007). Additional investigations on tissues from early postnatal rat offspring are presently under way.
Analysis of UV filters and other contaminants in human milk
In order to provide a basis for risk assessment, we further analyzed a number of UV filters, including 4-MBC and 3-BC, in human milk in three cohorts examined in 2004, 2005, and 2006. The chemical analyses were combined with a questionnaire on the use of a broad range of cosmetics. UV filters were present in over 75% of human milk samples, and a similar percentage of women reported use of one or more cosmetic products containing UV filters. UV filter levels were compared to levels of other classes of chemical contaminants, including PCBs, flame retardants (PBDEs), pesticides, fragrances, phthalate metabolites, and parabens, analyzed in the same milk samples (Schlumpf et al., Chemosphere 81: 1171, 2010). This is the first study providing realistic information on internal human exposure to cosmetic UV filters on a population basis.
(Funded by Swiss National Research Programme (NRP) 50, 5th EU Framework Programme (EURISKED/CREDO Cluster), Swiss Environmental Protection Agency (BAFU), Hartmann-Müller Stiftung, Olga Mayenfisch Stiftung).
Polybrominated diphenylethers (PBDE) have received increasing attention during recent years because their level was found to increase at an alarming rate in human and animal tissues, and because some congeners exhibited developmental neurotoxicity and estrogenic activity in vitro. We investigated developmental toxicity of the pentabrominated flame retardant PBDE 99 (meanwhile banned in the EU) in a rat model and observed effects on male puberty, male and female reproductive organs, brain and female sexual behavior at organ level and gene expression level (see Lichtensteiger et al., 2004, Ceccatelli et al., 2006, Faass et al., 2013). This project has been completed (funded by PBDE-NTOX, EU 5th Framework Programme).