EDC-MixRisk Annual Consortium Meeting will take place 22-24 May 2018 in Stockholm, Sweden. The programme will be packed with intense discussions preparing for the final year, as the project will end in spring 2019. The first day of the annual meeting will focus on discussing the key results and expected outcomes of the project, whereas the next two days will be dedicated to more in-depth discussions on the remaining tasks and plans for completing the work successfully.
Meet our EDC-MixRisk scientists working behind the scenes and learn more about their work. This time in line is Olle Söder who is Professor of Pediatrics and Head of Department of Women’s and Children’s Health, and of the pediatric endocrinology reproductive research unit, located at the Astrid Lindgren Children’s Hospital, Karolinska Institutet & University Hospital, Stockholm, Sweden. This is a translational research unit with a clinical division taking care of children with disorders of sex development (DSD) and other endocrine disorders. One of Prof. Söder’s major research interests is the impact of EDCs on testicular function.
Our group is working on the effects of EDCs on androgen production. Many recent epidemiological studies have shown an increased incidence of disorders of sexual development (DSD) in boys, such as cryptorchidism, micropenis and hypospadias. The background to this phenomenon is unexplained but one hypothesis is that exposure to environmental EDCs may contribute by interfering with critical effects of androgens during male fetal development. This hypothesis has previously been studied in animal models but investigations in humans are sparse albeit necessary due to species differences. Specifically, EDCs may affect the cascade of developmental events occurring in sexual differentiation of male fetuses by suppressing androgen production by fetal Leydig cells (FLC) or by disturbing proper organogenesis of external male genitalia. Such adverse action may ultimately result in DSD in newborn males.
We have exploited our unique access to human fetal tissue by pursuing studies in vitro with cells isolated from male fetal gonads and external genitalia. The aim of our part in EDC-MixRisk is to study EDC mixtures concerning their potential role in disturbances of male sexual development and, more specifically, in androgen production by human FLC and proliferation and differentiation of human fetal penial cells (hFPC) in vitro.
What have you discovered so far?
In EDC-MixRisk, we have the three health domains (growth and metabolism, neurodevelopment and sexual development). Our group has carried out studies with Mixture S0 to study the health effects on sexual development. The mixture reflects a “typically-measured” chemical mixture of EDCs in the SELMA mothers, and it thus corresponds to a real-life exposure situation.
We have found that “Mixture 0” at concentrations 10 and 100 nM significantly upregulated the expression of the important steroidogenic enzyme 5-alpha reductase 2 (SRD5A2) in human fetal penial cells (see fig.1). This suggests that phthalates in “Mixture 0” may affect the levels of the potent androgen DHT, which is critical for normal organogenesis of male external genitalia in humans. Studies on the other mixture, “Mixture 1” and its effects on human fetal penial cells and human fetal Leydig cells are currently ongoing.
What are the potential implications of your findings?
Our results suggest that “Mixture 0” has the potential to disturb the early fetal organogenesis of male external genitalia. This supports epidemiological evidence that environmental exposure of pregnant women to certain EDCs (e.g., phthalates) increases the risk for male fetuses to develop disorders of sexual development with genital abnormalities, such as micropenis, hypospadias and cryptorchidism. The ongoing analysis of “Mixture 1” will reveal if there is further support of the hypothesis that the development of male external genitalia in early gestation is sensitive to adverse actions of EDCs. The implications of such findings are of great importance as the results are derived from a model with human fetal tissue which is the direct target of EDCs in human fetal development.
Barbara Demeneix, Professor at the French National Museum of Natural History, is an internationally recognized expert on thyroid function and endocrine disruption. She is the work package leader of WP4 on identification of molecular initiating events in EDC-MixRisk project. Recently, she was interviewed for European Chemicals Agency’s newsletter on how chemicals can result in autism and IQ loss in developing children.
Endocrine disruptors are ubiquitous chemicals that interfere with body’s hormonal system, thereby producing harmful effects on human health including infertility, cancer and developmental disorders. These chemicals are released in our environment and can be found in daily products, such as in plastic bottles, toys, cosmetics, electronics, textile products and pesticides.
Exposure to these chemicals during foetal development poses a particular risk because the organ systems are still developing. Studies have shown that maternal levels of thyroid hormone during the first three months of pregnancy are determinant for the child’s brain development. Interference with the levels of maternal thyroid hormone will have a long-term impact on the formation and development of organs and neurons in the foetus. Many chemicals to which we are exposed can interfere with thyroid hormone signaling.
There has been a sharp increase in neurodevelopmental and metabolic diseases in modern western countries over the last decades. There is a biologically plausible mechanism between endocrine disruptors and their interference with thyroid hormone signaling: “Combining the facts that first, maternal hypothyroidism increases the risk of autism and second, that exposure to many chemicals reduces circulating thyroid hormones provides a strong case”, Professor Demeneix explains.
There is a need for rigourous testing as well as appropriate regulation of endocrine disrupting chemicals. The number of chemicals placed on the market is continuously increasing, so we are racing against time. However, you can try to limit your exposure to such chemicals. “Eating organic food can reduce your pesticide exposure. Also, you should air your home regularly and it is also very important to avoid smoking, either actively or passively, since this can alter thyroid hormone levels in early pregnancy,” Professor Demeneix advises. Further recommendations are developed in her latest book Toxic Cocktail.
Prof. Demeneix’s interview in ECHA’s Newsletter, People and Perspectives: How chemicals can result in autism and IQ loss in developing children, February 2018, Issue 1: https://newsletter.echa.europa.eu/home/-/newsletter/entry/how-chemicals-can-result-in-autism-and-iq-loss-in-developing-children
Her interview is also available on YouTube: https://www.youtube.com/watch?time_continue=1&v=LgPlKXBw4G4
Toxic Cocktail: how chemical pollution is poisoning our brains has been published by Oxford University Press in 2017: https://global.oup.com/academic/product/toxic-cocktail-9780190260934?cc=fr&lang=en&
The research performed in EDC-MixRisk helps to expand our knowledge on mixtures of endocrine disrupting chemicals. Meet our EDC-MixRisk scientists and hear their story on what they are doing and why it matters. This time in line is Carl-Gustaf Bornehag who is professor in Public Health Sciences and head of Public Health Sciences at Karlstad University, Sweden. He is an environmental epidemiologist with a major focus on health risks related to endocrine disrupting chemicals. He is also an adjunct professor at Icahn School of Medicine at Mount Sinai in New York.
Our focus is on population based epidemiological studies. Our main study – the SELMA study – includes around 2,000 mother-child pairs followed from early pregnancy and birth up to school age. In EDC-MixRisk, we are addressing a broad spectrum of exposures during early life (pregnancy and infancy period) that are significant for health and development during childhood in the three different health domains (sexual development, neurodevelopment, metabolism and growth). Our goal is to identify risk factors during sensitive periods of early life, e.g., environmental chemicals including endocrine disrupting chemicals, maternal stress, life styles etc. These risk factors can then be tested in controlled experimental studies in animal and cell models, which is our overall goal within the EDC-MixRisk project. Such interaction between epidemiology and experimental toxicology is a good platform for development of new and useful methods and tools for better risk assessment of harmful chemicals. The project brings together the experts from different disciplines and thus we have been able to develop good collaboration e.g. on risk assessment with Stockholm University and the group lead by professor Christina Ruden. We also have a major interest in exposure for mixtures of chemicals and have a deep collaboration with professor Chris Gennings and Icahn School of Medicine at Mount Sinai in New York which is also where I hold an adjunct professorship.
What preliminary results or interesting aspects have you discovered?
We have generated a lot of important data in the project. So far, some of the key results from our epidemiological efforts are:
– Prenatal exposure for phthalates (mainly from soft plastics) are associated to impaired sexual development in baby boys.
– Prenatal exposure for per fluorinated compounds (PFAAs) is associated to a lower birth weight with some differences between girls and boys.
– Prenatal exposure for mixtures of endocrine disrupting chemicals and the association to health effects in different domains.
– In terms of exposure, PVC flooring material is a strong source for human uptake of phthalates in pregnant women.
What are the potential implications of your findings and their usefulness for society?
I think that our epidemiological findings from the SELMA study may bring results of interest to the society, not least regarding the problem with mixture exposure where there is much more results to come but also clear needs for further research efforts and policy actions. However, I am more and more convinced that it is when we are putting our epidemiological efforts into a broader context – i.e., into an interaction with experimental studies and risk assessment activities- we will bring true societal impact and gains. Here we are just one part in a broader context, bringing one piece to the puzzle in the challenge to improve risk assessment of endocrine disrupting chemicals, mixtures and human health effects. And the main platform for this interaction is Swetox, collaboration between the Swedish universities, and of course all our international collaborators in this EC funded research project.
Exposure to EDCs is a potential threat to female reproductive health. Mixtures of chemicals can be found in serum of virtually every human being as biomonitoring studies have shown. Pregnant women are not an exception in this regard.
Levels of exposure to toxic chemicals and the resulting impacts on human health are largely determined by social as well as biological factors. Women, men, and children are exposed differently to toxic chemicals in daily life, including differences in the kinds of chemicals encountered as well as the level and frequency of such exposures. Furthermore, women and children vary in their physiological susceptibility to the effects of exposure to toxic chemicals.
EDC-MixRisk project addresses also the reproductive aspects to increase our understanding on the effects of chemical mixtures and gender on health. Dr. Pauliina Damdimopoulou’s group focuses on reproductive toxicology and the health effects of environmental endocrine active compounds. In EDC-MixRisk, the group will contribute to developing new methods and studying of health effects of chemical mixtures on women’s reproductive health.
Recently, the group has been working on the culture of a specific structure within the ovary, called a follicle. This follicle structure consists of supporting cells surrounding the growing egg. Previously, follicles have been successfully cultured using mouse tissue but challenges have remained in terms of human ovarian tissue.
The new model developed by Dr. Damdimopoulou’s group looks promising in applying the method to human tissue. Follicles in early stages of development are isolated from human ovaries that are obtained from Karolinska University Hospital Huddinge, and placed into a matrix to maintain their 3D structure. First, a cellulose matrix was tested but better success was achieved by using alginate which is a natural product of algae. The group is now working on improving the growth conditions to support follicle and egg development over a longer period of time.
A healthy human secondary follicle freshly isolated and ready for culture (Picture by Astrud Tuck)
The strength of this in vitro model is its direct relevance to humans. It also contributes to the replacement of animal-based methods and testing. Eventually, the research carried out by the group will increase our knowledge on how chemicals and chemical mixtures can affect women’s fertility. Furthermore, it is important to recognize the significant linkages between gender and chemicals.
For more information on the research team:
EDC-MixRisk project will organize a session “Integrating epidemiology and experimental toxicology to understand the risk of endocrine disrupting chemical mixtures on children” at the EUROTOX 2018 Congress which takes place in Brussels, 2-5 September 2018.
Endocrine disrupting chemicals (EDCs) and potential EDCs are found in various materials such as personal care products, pesticides, metals, additives or contaminants in food. A large body of evidence supports associations between exposure to chemicals and endocrine disrupting effects, leading to disorders in humans and wildlife. There is a need to handle EDCs according to the risks they pose, as single chemicals or as mixtures. This involves selection, refinement and development of tools for risk assessment of EDC mixtures to bring current procedures to a level where they can support proper risk management.
The session will give examples on how to determine and assess the risk for multiple adverse health outcomes based on molecular mechanisms involved, after early life exposure to complex mixtures of endocrine disrupting chemicals (EDCs). The session will present a new approach for identification of mixtures of EDCs that are associated with multiple adverse health outcomes in three health domains (growth and metabolism, neurodevelopment and sexual development) using epidemiological pregnancy cohort studies and via the application of novel and advanced bio-statistical methods to identify and prioritize chemical mixtures of concern. Experimental investigations in animal and cell models have thereafter been used in the project to test the mixtures in order to uncover molecular mechanisms underlying the effects seen in the epidemiological studies.
Furthermore, mixture effects on mesenchymal stem cell differentiation in relation to low birth weight and obesity later in life will be discussed at the session, as well as the transcriptomic and epigenomic profiling of human embryonic stem cells (hESC) during differentiation into neural progenitors exposed to EDC mixtures. The interdisciplinary approach, integrating epidemiology and experimental evidence, will facilitate assessment of risk and contribute to better risk management for EDCs and their mixtures.
EUROTOX 2018 homepage: http://www.eurotox-congress.com/2018/
EDC-MixRisk will organize its Steering Committee meeting in Milan, Italy, 12-13 December 2017. The meeting will be hosted by the Italian project partner, European Institute of Oncology (IEO), and it will gather all Principal Investigators of the project to discuss the overall progress and potential challenges. Particularly, the purpose of the meeting is to discuss the results generated in the project so far, to review the status of the tasks agreed in the Grant Agreement, as well as to plan and coordinate the work for the remaining period.
Prior to the meeting, EDC-MixRisk will be represented in a workshop on IPCHEM’s role on supporting the assessment of chemical mixtures. IPCHEM, Information Platform for Chemical Monitoring is the European Commission’s reference point for searching and accessing chemical occurrence data in Europe. The workshop is organized by Joint Research Centre in Ispra, Italy.
Meet our EDC-MixRisk scientists working behind the scenes and learn more about their work. This time Maria Jönsson is in the spotlight. She is an assistant professor, group leader and PI in the Uppsala University, Environmental Toxicology unit. The primary objective of her research group is to examine developmental effects of chemicals in vertebrates in vivo, including identification of molecular targets and mechanisms of action. Dr. Jönsson is a WP Leader in Work Package 5 (identification of adverse outcome pathways) in EDC-MixRisk.
The chemicals studied in EDC-MixRisk are mixtures of common chemicals that have been detected in serum in pregnant women and associated with risk for adverse effects in their children. These effects are related to sexual development, metabolism and growth, and neurodevelopment (S, G, and N mixtures, respectively). We are using two in vivo models (zebrafish and chicken) to examine how early exposure to the S0 and G0 mixtures affect sexual development and metabolism. More precisely we are looking at effects on development of reproductive organs and adipose tissue by the mixtures. We are also studying how the mixtures affect molecular pathways that are important for cell fate and cell turnover (Wnt/β-catening-signaling and p53/apoptosis) in zebrafish embryos.
What have you discovered so far and what your first results indicate?
We found that embryonic and larval exposure to the first growth mixture (G0) increased the number of adipocytes in zebrafish larvae. The same mixture reduced Wnt/β-catenin signaling in zebrafish embryos and preliminary data indicate that it also increases apoptosis in these embryos. These results indicate that G0 affects cell survival and cell fate in vivo and that one cell type in target is the adipocyte (its precursors). Our group did not find any effect of S0 on sex organ development in zebrafish or chicken.
What are the potential implications of your findings?
Our results suggest that ubiquitously present chemical mixtures have the capacity to stimulate adipogenesis (the process of fat cell differentiation) in humans and in wildlife at realistic exposure levels. The results are in line with in vitro results and studies conducted by other partners in the project so far. This is highly interesting in view of that the G0 mixture was composed of chemicals that were measured from the samples of pregnant women, and these chemicals correlate with low birth weight in the SELMA cohort. Low birth weight is associated with an increased risk for metabolic disorders.
PhD student Iuliia Savchuk, who is involved in the EDC-MixRisk project, defended her thesis 13 October 2017 in Astrid Lindgren´s Children Hospital, Stockholm.
The title of her thesis is “Diverse effects of endocrine-disruptive chemicals on Leydig and adrenocortical cell steroidogenesis in humans and rodents”. The aim of the thesis was to examine the impact of selected endocrine-disruptive chemicals (EDCs) on hormonal function of steroidogenic cells from mice and human.
EDCs have the potential to interrupt a normal steroidogenesis and thus result in harmful effects on reproductive health of humans and other organisms. Several previous studies have demonstrated a considerable decrease in fertility biomarkers such as sperm counts, and increase in malformations and undermasculinization of the male reproductive tract of mammals including humans.
The results of this PhD study indicated that environmental endocrine disruptive chemicals have diverse effect on androgen production in steroidogenic cells in rodents and humans. Bisphenol A and MEHP should be taken into consideration as possible causes of premature maturation in boys. Also, based on the results, it is recommended that pregnant women at an early gestational stage should try to avoid taking resveratrol due its ability to suppress steroidogenesis in the human fetal adrenals.
Reference information for the published papers:
- Savchuk I, Söder O, Svechnikov K (2013) Mouse Leydig cells with different androgen production potential are resistant to estrogenic stimuli but responsive to bisphenol A which attenuates testosterone metabolism. PLOS ONE 8 (8): e71722
- Savchuk I, Söder O, Svechnikov K (2015) Mono-2-ethylhexyl phthalate stimulates androgen production but suppresses mitochondrial function in mouse Leydig cells with different steroidogenic potenti Toxicol. Sciences 145 (1): 149-156.
- Savchuk I, Morvan M.-L, Søeborg T, Antignac J.-P, Gemzell-Danielsson K, Le Bizec B, Söder O, Svechnikov K (2017). Resveratrol inhibits steroidogenesis in human fetal adrenocortical cells at the end of first trimester. Molecular Nutrition & Food Research 61(2) DOI 10.1002/mnfr.201600522*.
- Savchuk I, Morvan ML, Antignac JP, Gemzell-Danielsson K, Le Bizec B, Söder O, Svechnikov K (2017) Androgenic potential of human fetal adrenals at the end of the first trimester. Endocr Connect. 6 (6): 348-359 doi: 10.1530/EC-17-0085.
We are exposed to multiple chemicals that make their way into the environment through several routes. Hormonal disruptive effects of many of these chemicals have been reported in experimental and/or epidemiological studies. Most of these disruptive effects have, however, been reported as an individual chemical rather than a mixture.
Research group led by Prof. Barbara Demeneix (France) in the EDC-MixRisk project has been tasked to identify and characterize the thyroid disrupting effect of endocrine disruptive chemical (EDC) mixtures using Xenopus laevis (an amphibian model for studying thyroid hormones, THs). These mixtures were shown within another work package to affect brain development (mix N0) and metabolism (mix G0) in humans.
The Xenopus Embryonic Thyroid Assay (XETA) was used to identify mixtures effect on thyroid signalling disruption. XETA was developed ten years ago by Prof. Demeneix and is currently being validated within the test guideline process at the OECD. The two mixtures suspected to disrupt either neurodevelopment or metabolism in human were found to disrupt TH signalling (at concentrations found in human concentration). Gene expression analysis of key genes in thyroid hormone signaling further confirmed TH disruption after mixture exposure. Finally, a phenotypical effect on mobility was reported as it was observed that all concentrations tested affected the light-induced movement of tadpoles.
A new research approach was applied, confirming experimentally the epidemiologically based hypothesis about these worrisome chemicals. It was shown that the two mixtures, N0 and G0, disrupted thyroid hormone signalling and adversely affected gene expression and mobility in amphibians. This amphibian model is relevant for thyroid disruption studies as metamorphosis is a highly thyroid hormone dependent process.
A matter of concern
Thyroid hormones are essential for normal brain development. A complete lack of thyroid hormone induces cretinism but a slight change in maternal thyroid hormone during pregnancy has also been shown to affect IQ and brain structure in children (Korevaar et al., 2016). Given this crucial role of thyroid hormone during early brain development, a correlation with N0 mixture effect is not surprising. Moreover, thyroid hormones play a critical role on metabolism, both peripherally and centrally. Therefore, it wasn’t unexpected that the mix G0 disrupted thyroid hormone signaling.
Currently, there is a sharp increase in neurodevelopmental and metabolic diseases in modern western countries. For example type II diabetes incidence doubled in the last 10 years while in US, 1 out of 42 boys were diagnosed autistic in 2016 (1 out of 5000 in 70’s). This increase is regardless of changes in diagnostic methods or protocols and access to better healthcare. Furthermore, in France, a 3% increase in congenital hypothyroidism has been observed for the last 20 years with exactly same diagnostic threshold (Barry et al. 2016). This gives importance to the environmental hypothesis on the increase in neurodevelopmental disorder incidence rate.
Best experimental practices and new methods
EDC-Mix Risk project offers a new strategy in risk assessment. Conventionally, compounds of concern are identified and a battery of in vitro or in vivo assays are conducted in order to extrapolate the human risk assessment. For endocrine disruptor risk assessment specifically, this strategy is extremely difficult. First, one should provide a burden of evidence, e.g. proof of exposure, adverse effects and a proof of mechanism of action. Second, the fact that we are exposed to a mixture of compounds makes the legislation process slow and difficult to handle.
In EDC-MixRisk, proof of exposure and correlation with adverse outcome are done on the basis of real-life exposure, derived from epidemiological data. Mechanism of action is provided by in vitro and in vivo assays such as the before mentioned the Xenopus Embryonic Thyroid Assay. New regulations through new data, protocols and approaches developed within EDC Mix-Risk could provide for safer chemicals for our next generation.
Barry Y, Bonaldi C, Goulet V, Coutant R, Léger J, Paty AC, Delmas D, Cheillan D, Roussey M. Increased incidence of congenital hypothyroidism in France from 1982 to 2012: a nationwide multicenter analysis. Ann Epidemiol. 2016 Feb;26(2):100-105.e4. doi: 10.1016/j.annepidem.2015.11.005. Epub 2015 Dec 12. PubMed PMID: 26775052.
Korevaar, T. I. M. et al. (2016) ‘Association of maternal thyroid function during early pregnancy with off spring IQ and brain morphology in childhood: a population-based prospective cohort study’, Articles Lancet Diabetes Endocrinol. Elsevier Ltd, 4(4), pp. 35–43. doi: 10.1016/S2213-8587(15)00327-7.