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.

 

Hi Maria – What your research group is studying in the experimental module of 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:

  1. 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
  2. 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.
  3. 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*.
  4. 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.

 

References:

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.

 


Phthalates are widely used in industrial and personal care products, including fragrances, shampoos, soaps, plastics, paints and some pesticides. Phthalates belong to a highly abundant group of semi-persistent EDCs that have been associated with reproductive dysfunction in humans, particularly in males. The anti-androgenic and / or anti-estrogenic effects of different phthalates esters have been shown both in in vivo and in vitro experimental systems.

However, studies examining the effects of simultaneous exposure to different phthalates, as happens in the real life, are very limited. In a mother-child cohort study conducted with partners of the EDC MixRisk project (the SELMA study), increased concentrations of a mixture of phthalates in the serum and urine of the mothers at the 1st-2nd trimester of pregnancy were associated with alterations in the anogenital distance (AGD) of the boys.

The research group at the University of Athens, led by Prof. Efthymia Kitraki, has focused on the effects of prenatal exposure to a phthalate mixture (mix S0) on the reproductive system, using the mouse as a model organism. The composition of the mixture S0 was derived upon combining epidemiological data from the SELMA cohort with advanced statistical analyses done by EDC-MixRisk Partners.

The female dams were exposed during the whole pregnancy to 4 different doses of mixture S0. Based on a mouse kinetic study, the 4 different doses of mixture S0 corresponded to x0.2, x1, x10 and x50 of the levels detected in the serum of mothers in the SELMA study. The litter size and sex ratio, the offspring body weight and anogenital distance (AGD) were examined at postnatal day 1. AGDs were also monitored till adulthood. Offspring were sacrificed at weaning and in adulthood.

The first results show that the AGD length (an indicator of in utero androgen concentration) was reduced in male offspring at postnatal day (PND) 21 and this was accompanied by increased number of abnormal testicular tubules at the same age. In the adult testes, the sperm content in the tubules was decreased and the aromatase gene expression was increased, the effects being more severe at the higher dose. Abnormal follicle development was also detected in the ovaries of the exposed offspring. Some of the aforementioned effects have been reported in the literature in studies using higher doses and individual components of the mixture S0. The analysis of the hormonal and molecular changes continues, but the present observations strengthen the significance of the overall EDC-MixRisk study design and the relevance of the mixture S0 composition as well as give cause for concern for adverse effects of EDCs.

EDC-MixRisk research group at University of Athens (from left to right): Emily Panagiotidou, Antonis Stamatakis, Lydia Panagopoulou, Efthymia Kitraki, Anastasia Repouskou

 


National Institute for Health and Welfare in Finland, THL, has analysed persistent organic pollutants (POPs) from samples of the two large pregnancy cohorts, SELMA and Life Child. POPs are organic chemical compounds which bioaccumulate in animals and humans. They include both 1) intentionally produced chemicals that are currently or were once used in agriculture, disease control, manufacturing, or industrial processes; and 2) unintentionally produced chemicals that are a result of some industrial processes or combustion.

THL’s chemical analysis group has measured 22 persistent organic pollutants (POPs) from 2373 SELMA serum samples from Sweden and 331 LIFE Child serum samples from Germany. Results of these samples have been used in statistical analysis to define which ones of these POPs are so-called “bad actors”, i.e. which ones are associated with adverse health effects addressed in this project. Those that were associated (hexachlorobenzene and p,p’-DDE) were added to experimental mixtures in concentration proportions that appeared in the serum samples of the pregnant women. The experimental mixtures will be further used in different cell and animal experiments to study their effects on sexual development, neurodevelopment and growth and metabolism.

Figures below show the comparison of HCB and p,p’-DDE in Swedish and German serum samples.

Figure 1. Mean HCB concentrations in SELMA (SWE) and LIFE Child (GER) grouped by sampling years and age of the mothers.

Figure 2. Mean p,p’-DDE concentrations in SELMA (SWE) and LIFE Child (GER) grouped by sampling years and age of the mothers.

Hexachlorobenzene (HCB) has previously been used as pesticide and small amounts are still also formed as by-product of industrial processes and combustion, whereas p,p’-DDE is the major metabolite of classical pesticide p,p’-DDT. The use of both HCB and p,p’-DDT is banned under the Stockholm Convention but limited use of p,p’-DDT in disease vector control continues, because of its effectiveness in reducing malarial infections, balanced by environmental and other health concerns.

 


Prof. Wieland Kiess, a partner in EDC-MixRisk project, is one of the main authors of the recently published research paper which has brought attention to the issue of Endocrine Disruptive Chemicals (EDCs) in Germany. The article, entitled in English “Endocrine disruptors: Evidence from epidemiological studies necessitates a critical review of model systems”, was published in a Federal German Health Journal, Bundesgesundheitsblatt.

The paper addresses adverse health effects of EDCs and depicts the epidemiological findings for EDC-caused effects in the fields of growth and metabolism, neurocognitive development and sexual development and reproduction. It also discusses the clinical importance of epigenetic changes caused by the action of EDCs during vulnerable phases of development.

Prof. Kiess says: “Epidemiological studies are often criticized because their reproducibility is not always guaranteed, but they remain the method of choice for the development and analysis of suitable model systems. Positive associations, in spite of sometimes conflicting results, are key in the selection of factors that can then be analysed in model systems in an unbiased way.”

Furthermore, the article highlights the need for closer linkage between epidemiological studies and mechanistic research into model systems, especially focusing on the interaction of different EDCs and the consequences of prenatal and early life exposure.

The original article was published in German. The abstract is available in English via this link.

Wieland Kiess is professor of paediatrics and director of the Hospital for Children and Adolescents of the University of Leipzig since 1998. He is PI of the German LIFE Child study cohort and actively involved especially in the project’s Work Package 2
, Epidemiology and biostatistics. His main research interests include chronic diseases in early life, interaction between environment, genes and society.


EDC-MixRisk was represented by Dr. Mattias Öberg (Karolinska Institutet/Swetox) at the WHO Chemical Risk Assessment Network meeting that was organized in Parma, Italy 20-22 June 2017.

The meeting, hosted by the European Food Safety Authority (EFSA), assembled 83 top organizations within the field of chemical risk assessment from all regions world wide. Development of a capacity building strategy was a major focus of the meeting, and other themes addressed included combined exposures to multiple chemicals, human biomonitoring, identifying new and emerging risks and new scientific approaches for regulatory safety assessment.

Furthermore, a new WHO publication titled “Chemical Mixtures in Source-water and Drinking-water” was launched at the meeting. The document provides an overview of available tools and practical recommendations to support the assessment and management of risks to human health associated with chemical mixtures in drinking-water and its sources.

WHO/IPCS also presented a report on public health impact of chemicals (2016), according to which it is estimated that 1.3 million lives and 43 million disability-adjusted life-years were lost in 2012 due to exposures to selected chemicals. However, data are only available for a small number of chemical exposures and people are exposed to many more chemicals every day.

A week before the network meeting, the issue of chemical health and environment was recognized at the highest political level. The Sixth Ministerial Conference on Environment and Health recently adopted the Ostrava Declaration (15 June 2017, Ostrava), aiming to e.g. promote actions on “minimizing the adverse effects of chemicals on human health and the environment; reducing the exposure of vulnerable groups to hazardous chemicals, particularly during the early stages of human development; strengthening capacities for risk assessment and research to secure a better understanding of human exposure to chemicals and the associated burden of disease; and applying the precautionary principle where appropriate”.

WHO Chemical Risk Assessment Network is a voluntary collaborative initiative whose overall goal is to improve chemical risk assessment globally through facilitating sustainable interaction between institutions on chemical risk assessment issues. The Network has been established to enhance global efforts to assess risks to human health from exposure to chemicals.

More information about the WHO network and its activities:
http://www.who.int/ipcs/network/en/


Meet our EDC-MixRisk scientists and hear their story on what they are researching and why it matters. Joachim Sturve is assoc. professor and lecturer at the Department of Biological and Environmental Sciences at the University of Gothenburg, Sweden. He is an ecotoxicologist and the main focus of his research is on the effects of pollutants in aquatic organisms, primarily fish.


Hi Joachim – What your research group is researching in the experimental module of EDC-MixRisk?

We are studying the effects of EDC mixtures in fish, primarily zebrafish. The zebrafish genome matches approximately 70% to human orthologs, and they serve as a model species for biomedical research. Our work in EDC-MixRisk is divided into two parts. First, acute exposures with the aim to identify modes of action and possible biomarkers for this kind of exposures. Second, long-term transgenerational exposures with the aim to find epigenetic effects of EDC mixture. F0 generation of fish has been fed with low levels of EDC mixtures from larval stage until sexual maturity. Epigenetic alteration will be studied in the F1 and F2 generations.

 

What have you discovered so far and what your first results indicate?

The short turn acute studies have generated a lot of fascinating results and it is evident that the thyroid system is affected by these mixtures. We have identified specific components (genes) in the thyroid system which might be interesting for future studies. Epigenetic alterations in F1 and F2 generations will be studied during the fall of 2017.

 

What are the potential implications of your findings in a bigger picture?

In EDC-MixRisk, the various cell and animal models will be applied to epidemiological findings from two independent human cohorts. This gives a unique opportunity for collective data analysis comparing the reliability of experimental models in reproducing human health effects. Our results then again contribute to adding one more piece of the puzzle when it comes to EDCs effects on the thyroid system. They will hopefully also generate possible biomarkers for future screening and monitoring, which is relevant for risk assessment.

 


EDC-MixRisk will organize a workshop in Brussels, 25-26 September 2017. The aim of the workshop is to discuss the Adverse Outcome Pathway (AOP) approach and to explore the approach from the EDC-MixRisk perspective. The second day of the workshop will focus especially on aspects of societal impact and building key messages based on recent research results.


The EDC-MixRisk Consortium came together for its annual meeting in Stockholm, 15-17 May, to discuss the project’s progress and future activities.

The meeting was organized as a three-day event, combining an 1) Adverse Outcome Pathway  (AOP) workshop 2) a joint meeting between EDC-MixRisk and EDC-2020 to present scientific results and 3) one and half days dedicated for the EDC-MixRisk Consortium discussions.

The meeting was a great opportunity to learn more about an AOP approach and get highlights and glimpses of scientific results generated within the two projects, EDC-MixRisk and EDC-2020. Furthermore, the meeting provided a great forum to share lessons learned as well as to receive feedback and to discuss further ideas and plans with the Members of the Scientific Advisory Board.

The programme of the last one and half days of the meeting gave an overview of the project progress and on the tasks within the different modules. Also an update on management and dissemination activities was presented. The module breakout sessions drilled into the challenges, future plans and details of the work which were reported on the final day of the meeting.

The first results generated within the project have demonstrated the validity of project’s integrated approach – interaction between epidemiology and experimental toxicology, and the need to take mixture effects into account for risk assessment. It was summarized that overall, the project is progressing well and is largely on track in terms of its tasks and timeline.

The 2018 Consortium meeting will be held again in Stockholm, the hometown of the Project Coordinator, Prof. Åke Bergman.

 

 

 

 

 

 

EDC-MixRisk is a four-year project financed through the EU Commission’s programme Horizon 2020, which started in the spring of 2015. It involves participation from six Swedish Swetox-universities, as well as five European and one American. The project focuses on the effects of mixtures of endocrine disruptive chemicals (EDCs) on children.

The interdisciplinary and integrated research approach of EDC-MixRisk includes:

  • epidemiology of two child cohorts focused on three health domains (growth and metabolism, neurodevelopment, and sexual development)
  • a web of experimental toxicology and molecular biology approaches to determine multiple adverse health outcomes with pathways starting at the molecular level during early life exposure to EDC mixtures
  • regulatory toxicology for development of a transparent and systematic risk assessment framework. Together this interdisciplinary research, integrating epidemiologic and experimental evidence, will facilitate assessment of risk and societal impact ensuring better risk management for EDCs and their mixtures.

 

EDC-2020 is a five-year research program on endocrine disrupting chemicals financed by approx. one million euro a year by the Swedish Research Council Formas on behalf of the Swedish Government. The core of the project is five research areas and a training program. Eleven Swedish Swetox-universities have joint together in the efforts of EDC-2020.

EDC-2020 aims to:

  • create an enabling environment for strong research on EDCs and for scientific advances and innovations, which will benefit the field of toxicology-related sciences as a whole.
  • establish an overarching national program for research on EDCs, optimizing cooperation between national universities as well as with the Swetox facilities in Södertälje, and improve interdisciplinary research.
  • establish an international EDC collaboration platform including world-leading experts and to link students in toxicological sciences across the country.