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Periodic Report (September 2016) XENROID 329996
Revised version 2
1. PUBLISHABLE SUMMARY
The objective of this project is to investigate environmental endocrine-disrupting
chemicals posing a threat to wildlife. Thyroid and steroid hormone systems are used
to describe endocrine-disruption. The first outgoing year comprised of developing and
establishing chemical analysis and gene expression methods enabling assessment of
endocrine health in wildlife. The second outgoing year focused on controlled in-vitro
and in-vivo toxicology exposure studies of suspected endocrine disruptors.
During this project we established; highly sensitive mass spectrometry assays
to determine a) thyroid hormones and new metabolites, b) corticosteroids, c) selected
xenobiotics and metabolites. Gene expression assays quantifying selected genes
important for the steroidogenic pathway (CYP17 and CYP19) at mRNA level in
amphibians. The amphibian in-vivo model Xenopus laevis was used to study the
impact of pesticides on wildlife, and to clarify endocrine-disruption mechanisms and
potential cross-talk among biomarkers. A fungicide, named tebuconazole, was found
to bioconcentrate in adipose tissue, but also in kidney and brain in the adult male
frogs. Importantly, we discovered the fungicide was an endocrine-disrupting chemical
by suppressing the action of essential of enzymes important for steroid biosynthesis;
resulting in altered androgenic and estrogenic hormone levels.
Establishing new scientific methodologies will assist in deeper mechanistic
understanding and, compared to physiological measures, may give earlier warnings of
endocrine-disruption in wildlife. Targeting the vital thyroid hormone synthesis, we
developed such a methodology to determine 11 different hormones and metabolites in
amphibian studies. The extensive work resulted in use of novel isotopic-dilution mass
spectrometry, and was applied on two frog species; adult Xenopus laevis and tadpole
Rana catesbeiana. We found thyroxine as dominant thyroid hormone in adult frogs,
however we found previously un-described thyroid hormone metabolites in tadpoles.
We are in the process of applying this state-of-the-art scientific tool in a number of
studies.
This grant enabled me to establish a sound scientific collaboration and
research network with leading researchers, societies and institutions in North America.
The expected results from my work and collaboration will impact the research within
endocrinology and endocrine-disruption. Continuing work and final results will focus
on assessing new endocrine-disruptors and investigate potential cross-talk between
thyroid and steroid endocrine axes.
http://pharmacy.ku.dk/research/section_analytical_biosciences/toxicology_lab/xenobi
otic_induced/
2. PROJECT OBJECTIVES FOR THE PERIOD
The overall project objective is to investigate xenobiotic endocrine disruptors in
wildlife. The first outgoing year comprised of developing and establishing chemical
analysis and gene expression methods enabling assessment of endocrine health in
wildlife. The second outgoing year focused on controlled in-vitro and in-vivo
toxicological exposure studies of suspected endocrine disruptors. Much data have
already been processed and this work is continuing.
The key project objectives for the period were;
i.
Development and establishment of chemical and gene expression tools.
I managed to develop new chemical and gene expression tools. These
tools are published in two scientific papers (listed below). More work is
continuing within this field and additional project publications are
expected within 1-2 years.
1) Poulsen, R., Luong, X., Hansen, M., Styrishave, B., Hayes, T.,
2015. Tebuconazole disrupts steroidogenesis in Xenopus
laevis. Aquatic Toxicology 168, 28–37.
2) Hansen, M., Luong, X., Sedlak, D.L., Helbing, C.C., Hayes,
T., 2016. Quantification of 11 thyroid hormones and
associated metabolites in blood using isotope-dilution liquid
chromatography tandem mass spectrometry. Anal Bioanal
Chem 408, 5429–5442.
ii. Identification of endocrine disrupting chemicals and elucidation of cross-talk.
Here, I attempted to develop and implement an in-vitro thyroid hormone
cell-line. After many problems we decided to implement an improved and
established thyroid hormone producing in-vitro system. Experimental data
are proceeding and several publications describing endocrine disrupting
chemicals and cross-talk are expected over the years to come.
iii. Wildlife cohort and in-vivo study.
A number of amphibian in-vivo studies investigating endocrine disrupting
chemicals have been done and this collaboration with UC Berkeley is
continuing. In addition, I have established collaboration with University
of Victoria for future studies.
I have secured a number of wildlife samples to assess potential endocrine
imbalance. In addition to sea lion and polar bear, I secured killer whale
and beluga whale (all with appropriate ethical approval and permits). This
work is continuing, however the first study using beluga whales will be
submitted for scientific peer-review shortly.
3. WORK PROGRESS AND ACHIEVEMENTS DURING THE PERIOD
Summary of progress towards objectives. As outlined in the work plan (cf. Annex
I), we aimed to 1) develop chemical and gene expression tools, as well as 2) identify
new endocrine-disrupting chemicals and elucidate cross-talk, and finally 3) collect
wildlife samples and perform controlled in-vivo studies.
We have successfully established 1) chemical and gene expression tools; i.e.
highly sensitive mass spectrometry assays to determine a) thyroid hormones and
metabolites, b) corticosteroids, c) selected xenobiotics and metabolites, and d) gene
expression assays quantifying selected genes at mRNA level in amphibians. We have
2) identified new endocrine-disrupting chemicals and elucidated cross-talk; i.e.
discovering the fungicide tebuconazole as an endocrine disruptor. We have 3)
collected wildlife samples and perform controlled in-vivo studies; i.e. collected
biological material from killer whales under two Arctic expeditions and performed
several amphibian in-vivo investigations.
We encountered problems when sampling wildlife and when developing invitro thyroid hormone cell-line. During first Arctic expedition it was only possible to
obtain samples from killer whale and not polar bear. It has been more difficult to
sample polar bear, however it was possible to obtain 10-20 killer whale samples in
2014. Killer whale is the apex predator in the marine environment, and contains even
higher xenobiotic body burden compared to polar bear. Consequently, replacing polar
bear with killer whale is reasonable. However, through my established collaboration
with Norwegian and Danish researchers it will be feasible to obtain biological
samples from polar bear the years to come. Moreover, we encountered problems
when establishing a planned in-vitro thyroid hormone cell-line (using thyroid gland
tissue). We successfully implemented an improved and more robust thyroid hormone
producing in-vitro enzymatic system.
Summary of progress towards researcher training. During my stay at UC
Berkeley I have received intense training in mass spectrometry and bioanalysis from
out-going scientist in charge (Prof. David Sedlak) and collaborator Prof. Tyrone
Hayes. In addition, I have trained under-graduate and graduate students in using the
newly established assays, and performed outreach activities (oral presentations and
posters) at scientific conferences and meetings. Moreover, I have initiated scientific
collaborations with Prof. Caren Helbing, University of Victoria, BC, Canada and Prof.
Martinovic-Weigelt, University of St. Thomas, MN, USA focusing on amphibian and
fish toxicology, respectively.
Use of resources. Resources, as researcher-months, are neither in excess nor a
limitation. Therefore, timeframe and resources are following the original outlined
work plan. Other resources have mainly covered wet lab materials. We obtained cofunding from a private foundation (the Carlsberg Foundation, €35,000) to cover
expenses for two Arctic field expeditions.
4. ADDITIONAL INFORMATION
It was mentioned some relevant ethics authorization should be provided covering the
period from 01/07/2014.
All required ethics authorizations were obtained. The biological samples from
marine mammals at MMC were collected before the actual ethical permit expired (30
June 2015, attachment NOAA-Permit.PDF). Therefore, no additional permit is
required. Furthermore, we obtained an additional ethical permit to expand our
investigations on beluga whales (NOAA-Permit 2.PDF). Scientific publications are
outlined based on these data.
5. DISSEMINATION ACTIVITIES
We outlined several dissemination activities (cf. Annex I), this includes 1) Marie
Curie Ambassador, 2) public talks, 3) workshops, 4) non-specialist publication of
results and 5) multimedia.
1) I have encouraged young European researchers at conferences and workshops
(lately at the Society of Environmental Toxicology and Chemistry annual conference
in Europe, May 2016) to apply for Marie Skłodowska-Curie actions research
fellowships. In addition, I have been involved in scientific advising teachers at
Berkeley High school. 2) I made public talk platforms with faculty staff at UC
Berkeley, presenting this project its findings and outcomes. 3) I have participated in
several conferences and workshops; especially the Workshop in The Endocrine
Society at Penn State College of Medicine and the Society of Environmental
Toxicology and Chemistry in North America were fruitful and expanded my scientific
network considerably. 4) I envision non-specialist publication of results by the end of
the project. 5) A webpage has been launched, however I envision more multimedia
activities by the end of the project.
Executed activities. The research findings are published, or in manuscript form, in
renowned scientific journals, and presented at workshops and conferences. Abstracts,
presentations and full-length articles are attached (as PDF files) to this Periodic
Report.
a) Website:
http://pharmacy.ku.dk/research/section_analytical_biosciences/toxicology_lab
/xenobiotic_induced/.
b) Peer-reviewed journal papers:
1. Poulsen, R., Luong, X., Hansen, M., Styrishave, B., Hayes, T., 2015.
Tebuconazole disrupts steroidogenesis in Xenopus laevis. Aquatic
Toxicology 168, 28–37.
2. Hansen, M., Luong, X., Sedlak, D.L., Helbing, C.C., Hayes, T., 2016.
Quantification of 11 thyroid hormones and associated metabolites in
blood using isotope-dilution liquid chromatography tandem mass
spectrometry. Anal Bioanal Chem 408, 5429–5442.
c) Conferences and workshops
1. Society of Environmental Toxicology and Chemistry | Europe (May
2016)
2. University of Copenhagen, Faculty of Science (March 2016)
3. University of California Berkeley, Department of Integrative Biology
(June 2015)
4. Society of Environmental Toxicology and Chemistry | North America
(November 2014)
5. Workshop on Measuring Estrogen Exposure and Metabolism, The
Endocrine Society, Penn State College of Medicine (March 2014)
6. University of California, Berkeley, Department of Civil and
Environmental Engineering (September 2014)
Planned activities. My immediate research activities include implementing the
scientific tools trained at the out-going host. In addition, a number of scientific
dissemination activities are planned.
d) Scientific manuscripts in preparation for peer-review:
1. Revisiting thyroid hormone analysis: a review
2. Combinational endocrine effects of xenobiotics in amphibian model
3. Thyroid hormones and associated metabolites in beluga whale
(Delphinapterus leucas)
4. Uptake and incorporation of 14C-cholesterol in human adrenal
corticocarcinoma cells (H295R) and online radiodetection of in-situ
14
C-produced steroid hormones
5. Contaminants in East Greenland fish
6. ADHD medicines affect steroidogenesis: in-vitro study using H295R
cell line
7. The North Atlantic Killer whale is heavily affected by contaminants
e) Conferences and workshops:
1. Concluding XENROID workshop
2. Society of Environmental Toxicology and Chemistry
6. PROJECT MANAGEMENT
Project planning and status. Project status meetings have been made, according to
the work plan, monthly or bi-monthly with Out-going scientist in Charge Prof. David
Sedlak and in part with Prof. Tyrone Hayes and researchers at MMC.
Problems encountered. We forecasted early in the project, that there were
insufficient funds available for fieldwork to collect wildlife samples. We solved this
by applying for additional field-expedition co-funding from the Carlsberg Foundation.
Carlsberg granted some €35,000 to collect Arctic wildlife (grant no. CF14-0999).
Development of website. The website was established at the beginning of the project
period.
http://pharmacy.ku.dk/research/section_analytical_biosciences/toxicology_lab/xenobi
otic_induced/
Ethical issues. Appropriate permits were needed to perform in-vivo studies and to
collect wildlife samples. In-vivo studies were executed at Prof. Tyrone Hayes lab UC
Berkeley and at Prof. Caren Helbing University of Victoria. University of California
and University of Victoria animal care and use programs, protocols, and procedures
were followed and appropriate permits obtained. Similarly for wildlife samples
permits were obtained from Convention on International Trade in Endangered Species
of Wild Fauna and Flora, and the US Department of Commerce National Oceanic and
Atmospheric Administration (Attached to this report).
7. LIST OF REPORT ANNEXES (Attached as PDF files).
Article 1.PDF: Peer-reviewed journal article. Poulsen, R., Luong, X., Hansen, M.,
Styrishave, B., Hayes, T., 2015. Tebuconazole disrupts steroidogenesis in Xenopus
laevis. Aquatic Toxicology 168, 28–37.
Article 2.PDF: Peer-reviewed journal article. Hansen, M., Luong, X., Sedlak, D.L.,
Helbing, C.C., Hayes, T., 2016. Quantification of 11 thyroid hormones and associated
metabolites in blood using isotope-dilution liquid chromatography tandem mass
spectrometry. Anal Bioanal Chem 408, 5429–5442.
Website.PDF: Project website printout.
Presentation 1.PDF: Conference platform presentation at Society of Environmental
Toxicology and Chemistry | Europe (May 2016).
Presentation 2.PDF: Presentation at Society of Environmental Toxicology and
Chemistry | North America (November 2014).
Presentation 3.PDF: Public talk at University of California, Berkeley, Department of
Civil and Environmental Engineering (September 2014).
Presentation 4.PDF: Workshop on Measuring Estrogen Exposure and Metabolism,
The Endocrine Society, Penn State College of Medicine (March 2014).
Manuscript in prep 1.PDF: Peer-review article outline of “Thyroid hormones and
associated metabolites in beluga whale (Delphinapterus leucas)”.