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The Belgian Society for Toxicology and Ecotoxicology
Joint annual meeting, November 26, 2015
The safety of nanomaterials: opportunities & challenges
University of Antwerp, Klooster van de Grauwzusters, Stadscampus
Building S, Lange Sint-Annastraat 7, 2000 Antwerp, Belgium.
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The Belgian Society for Toxicology and Ecotoxicology
The safety of nanomaterials: opportunities & challenges
9:30 Registration
9:50 Welcome by the chairman
10:00 - 10:50 Ronny Blust (University of Antwerp, Belgium) "Fate and effects of nanomaterials in the environment"
10:50 – 11:30 Karin Wiench (BASF, Germany) “Nanomaterial risk assessment - an industry perspective”
11:30 – 12:00 Coffee break
12:00 - 12:10 Pitch presentations by the meeting sponsors: Nanowal, VITO & j.j.bos b.v.
12:10 – 12:30 Freya Joris (Ghent University, Belgium)
“Multiparametric high content imaging reveals distinct cell type-specific nanotoxicity profiles”
12:30 – 12:50 Sarah Deville (Flemish Institute for Technological Research & Hasselt University, Belgium)
“Safety characterization of gold nanoparticles in complex mixtures”
12:50 – 13:10 General assembly BelTox
12:50 – 14:00 Lunch and poster session
14:00 – 14:20 Virginie d’Ursel de Bousies (Université Catholique de Louvain, Belgium)
“Monocytic myeloid-derived suppressive cells as a new component of the carcinogenic response to carbon nanotubes”
14:20-14:40 Marie-Astrid Parent (Université Catholique de Louvain, Belgium)
“Early and sustained immunosuppressive macrophage response to carcinogenic carbon nanotubes in a rat mesothelioma bioassay”
14:40-15:00 Deniz Öner (KU Leuven, Belgium)
“Effects of genotoxicity, DNA methylation and transcriptional changes by Carbon Nanotube exposure in vitro”
15:00 – 15:40 Hans Bouwmeester (RIKILT, Wageningen University, The Netherlands)
“Gut-on-a-chip models for risk assessment of nanomaterials”
15:40 – 16:10 Coffee break
16:10 – 16:50 Raymond Schiffelers (University Medical Center Utrecht, The Netherlands) “Opportunities and safety of nanomaterials in medicine”
16:50 - 17:30 Evelien Frijns (Flemish Institute for Technological Research, Belgium) “Workplace exposure to inhaled nanoparticles”
17:30 – 17:45 Young scientist awards and closing by the chairman
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The Belgian Society for Toxicology and Ecotoxicology
Joint annual meeting, November 26, 2015
The safety of nanomaterials: opportunities & challenges
Meeting sponsors
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Sponsors
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The Belgian Society for Toxicology and Ecotoxicology
ABSTRACTS INVITED SPEAKERS
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10:00 - 10:50 Ronny Blust (University of Antwerp, Belgium)
"Fate and effects of nanomaterials in the environment"
Fate and effects of nanomaterials in the environment
Ronny Blust, Department of Biology, University of Antwerp, Belgium
The development and application of nanobased materials has grown strongly over the last years with applications
in very different areas such as cosmetics, clothing, electronics and many other industrial applications. The
nanomaterials obtain their specific characteristics and properties from their nanostructural organisation and
dimensions. The most important groups of nanomaterials are carbon based, metal based, dendrimer or composite
type of nanomaterials. Several nanomaterials are metals or metaloxides but with properties and reactivities that
are quite different from the composing metals. These specific characteristics have raised concern in relation to the
potential impact on human and environmental health. The increased production and application of the
nanomaterials in various products inevitably results in the release to the environment with unknown
consequences. The uncertainties associated with the fate and potential toxicity of nanomaterial accumulation in
the aquatic and terrestrial environment has triggered a number of research initiatives to fill in the large data and
knowledge gaps that exist on these issues.
Environmental risk assessment requires information on current and future exposure levels to nanomaterials and
identification of the most sensitive environmental compartments. Several of the nanomaterials show a complex
and dynamic behaviour including dissolution, transformation and aggregation reactions. Some of the nanomaterials
are known to be highly labile while others are much more persistent. So far most of the environmental fate studies
that have been performed are small scale laboratory based assessments and many of them lack environmental
realism both in terms of exposure scenarios and environmental conditions. Field measurements of nanomaterials
are virtually lacking and are also an analytical challenge. Nonetheless, such information together with experimental
fate studies and model simulations is needed to obtain realistic estimates of the exposure levels and forms in which
the nanomaterials are present (i.e. predicted exposure concentrations).
From an ecological perspective information is needed concerning the bioavailability, mode of action and
toxicological effects of the nanomaterials across species and levels of biological complexity. In vitro and in vivo
models are used to characterise the uptake and molecular response profiles of exposure to nanomaterials and this
has revealed a number of general, but in some cases also more nanospecific interactions and effects. For example,
the toxicological profile of a number of metal based nanomaterials is very similar to that of the free metal ion but
the internal compartmentalisation may be different if the particles remain relatively stable. Most of the
ecotoxicological information refers to acute toxicity studies and there is a need to document chronic effect levels in
a species sensitivity distribution based approach. Together with realistic estimates of exposure this information can
then be used to develop the ecological quality standards that are needed for risk assessment.
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10:50 – 11:30 Karin Wiench (BASF, Germany)
“Nanomaterial risk assessment - an industry perspective”
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15:00 – 15:40 Hans Bouwmeester (RIKILT, Wageningen University, The Netherlands)
“Gut-on-a-chip models for risk assessment of nanomaterials”
Gut-on-a-chip models for safety assessment of nanomaterials
Hans Bouwmeester, Richard Helsdingen, Anna K. Undas, Ruud J. Peters, Hans J. Marvin, Stefan Weigel, Meike van
der Zande
RIKILT – Wageningen University & Research Centre, 6700 AE Wageningen, The Netherlands.
The great challenge of safety assessment of nanomaterials is to keep pace with innovations in research and
development that is resulting in an ever increasing diversity of nanomaterials. In conventional risk assessment, in
vivo studies are still regarded essential. However, in vitro studies are of great value for mechanistic (adverse
outcome pathway) studies, and the establishment of nanomaterial key descriptors.
After a decade of nanosafety research no single nanomaterial metric has been identified that solely explains the
observed biological effects. Identification of key descriptors would greatly advance discussions on grouping and
read- across. For this not only cost efficient analytical tools need to be developed that describe nanomaterials in
relevant biological environments, but also advanced physiologically relevant in vitro models.
We have developed and implemented sensitive nanomaterial sizing methods for the detection of nanomaterials in
biological samples. I’ll show the applicability of single-particle-ICP-MS based techniques in combination with FFF
and HDC to improve the sensitivity of this method. As a next step, we are now developing procedures to assess the
solubility rate of nanomaterials, likely an important key descriptor.
Is the selection of the in vitro model important for the identification of a key descriptor? I’ll discus this using a
recent study in which we compared the effects of differently sized, highly characterized silver nanoparticles on
Caco-2 cells, as the most established model for the gut epithelium, and on MCF-7 cells. Using data from
nanomaterial characterization, imaging and toxicogenomic approaches I’ll show that while the mechanism of action
of silver nanoparticles may seem different in different cell lines, highly comparable biological pathways are used by
these cells.
Can the value of in vitro studies be increased by using advanced in vitro concepts? Gut-on-a-chip concepts are
being developed through innovative combinations of microfluidics chips and bio-engineering. Thus an environment
is created that both physically and physiologically simulates the in vivo microenvironment far better than current
static in vitro models. This, leads to more relevant models for kinetic absorption and effect studies. I’ll show data
on our gut-on-a-chip model based on Caco-2 cells. The chip constitutes three layers. The middle layer contains a
porous membrane on which the cells were grown. Both above and below the middle (membrane/cell) layer two
fluid flows are applied. The flow impacts the growth of the cells and we evaluated the morphological and
physiological properties of the gut-on-a-chip by comparison with a static Transwell® Caco-2 model.
Combining cost efficient analytical nanomaterials characterization tools, with advanced in vitro models will be of
great benefit for the identification of nanomaterial key descriptors to be used in innovative approaches for the
safety evaluation of nanomaterials.
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16:10 – 16:50 Raymond Schiffelers (University Medical Center Utrecht, The Netherlands) “Opportunities
and safety of nanomaterials in medicine”
Opportunities and challenges for nanoparticles in medicine
Raymond Schiffelers
Laboratory Clinical Chemistry & Hematology, University Medical Center Utrecht, Utrecht, The Netherlands
One of the most attractive applications of nanoparticles in medicine is their ability to change the
distribution of drugs in the body. By encapsulation or association of a drug with a nanoparticle, the tissue
accumulation is dictated by the characteristics of the particle rather than the characteristics of the free
drug. In this manner accumulation can be enhanced at the target site, whereas accumulation in toxicity-
sensitive tissues may be avoided.
In this presentation, I will use the liposome as an example of the promises and challenges that
nanoparticles face in the translation from laboratory to clinical application. Early studies with liposomes
during the 1970’s-1980’s pointed towards the remarkable capacity by which the cells of the mononuclear
phagocyte system, particularly in the liver and spleen, where able to recognize liposomes and clear them
from the circulation. This occurred despite the fact that they are composed of natural phospholipids and
cholesterol, a composition very similar to the cell membrane.
In the 1990’s, the capacity of coating the liposome surface with poly(ethyele glycol) was shown to
decelerate macrophage recognition and allowed accumulation at other sites in the body. The first
commercial formulation that benefited from this development was Doxil. This liposomal formulation of
doxorubicin was shown to reduce the cardiac toxicity of doxorubicin and increased accumulation of the
drug in the tumor. But due to the changed tissue distribution, a new toxicity became apparent: hand and
foot syndrome.
Current developments in the liposome filed include triggered release, where specific signals in the target
tissue (such as enzymatic activity) or exogenous triggers (like focused ultrasound) are used to destabilize
the liposome to improve control over drug release.
One of the most exciting developments in the past few years is the recognition of extracellular vesicles as
endogenous nanosized carrier systems. Recent studies point to these cell derived membrane vesicles as
carriers of RNA, proteins and other biomolecules over longer distances within the body. These vesicles
could form a source of inspiration for a new generation of drug delivery systems
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16:50 - 17:30 Evelien Frijns (Flemish Institute for Technology, Belgium)
“Workplace exposure to inhaled nanoparticles”
Workplace exposure to inhaled nanoparticles
Evelien Frijns
Flemish Institute for Technological Research (VITO NV), Environmental Risk and Health, Boeretang 200, 2400 Mol,
Belgium, Contact e-mail address: [email protected]
Engineered nano-objects (<100nm) and their agglomerates and aggregates (> 100nm) (NOAA) are handled today in
workplaces in diverse occupational environments from research to production to use and applications in work
processes. This presentation will give a brief overview of the current Belgian legislation towards the health
protection and safety of workers against the risks of NOAA at the workplace. Legislation requires a risk assessment
and thus a thorough understanding of the exposure potential for workers. The exposure potential can be
determined in real workplaces but also in simulated conditions. The metrics and monitors/samplers that are used
for these assessments will be illustrated.
Current methodology for conducting consistent exposure related measurements and assessments of aerosols
containing engineered NOAA in workplace operations will be further explained. No health based limit values have
been defined yet to compare the measurements results. The Netherlands proposed to use nano reference values
instead.
Current methodologies for Nanoparticle release studies under laboratory conditions (simulation) will also be
illustrated.
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The Belgian Society for Toxicology and Ecotoxicology
ABSTRACTS Oral presentations
Young Scientist Competition
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Multiparametric high content imaging reveals distinct cell type-specific nanotoxicity profiles
Freya Joris (a), Daniel Valdepérez (b), Stefaan J. Soenen (c), Stefaan C. De Smedt (a), K. Raemdonck (a)
a Lab of General Biochemistry and Physical Pharmacy, Ghent University, Ghent, Belgium.
b Department of Physics, University of Marburg, Marburg, Germany
c Biomedical MRI Unit/MoSAIC, KULeuven, Leuven, Belgium.
Freya Joris
While nanotechnology is advancing rapidly, nanosafety tends to lag behind since mechanistic insights into how
nanoparticles (NPs) can cause cellular injury remain rare. To tackle this issue, standardization of nanosafety
assessment is imperative. In this regard, we believe that the importance of rational cell type selection should not
be overlooked since the applicability of cell lines was recently questioned due to their altered phenotype in
comparison to the native cells. Hence, we evaluated the impact of the cell type selection on the outcome of in vitro
nanosafety evaluations. Hereto, we assessed NP-induced effects in a neuroblastoma cell line, neural progenitor cell
line and neural stem cells from both human and murine origin. Acute toxicity measurements revealed that the
neural stem cells were most affected by AuNPs, AgNPs and IONPs exposure while the mouse neuroblastoma cell
line was the least sensitive. Additionally, using a high content imaging approach, we exami ned the impact of IONP
exposure on the production of reactive oxygen species, intracellular free calcium, mitochondrial health and cell
morphology. Interestingly, we observed a cell type-specific combination of effects for each cell type that could not
be retrieved in any other cell type included in this study. This indicated that the IONPs impaired cellular
homeostasis via distinct mechanisms. In conclusion, our data clearly reveal cell type-specific toxicity profiles and
demonstrate that a cell line cannot simply be applied to model in vitro nanotoxicity. Therefore, we propose to
avoid non-human cell types, to identify a set of standard cell lines for screening purposes and to select the cell type
for detailed nanosafety studies based on the intended NP application and/or expected exposure.
Keywords: high content imaging, multiparametric, nanosafety, in vitro
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Safety characterization of gold nanoparticles in complex mixtures
Sarah Deville a,b; Birgit Baré a,c; Jordi Piella d,e,; Kristof Tirez a; Peter Hoet c; Victor F. Puntes d,g,h; Inge Nelissen a
a Flemish Institute for Technological Research, Mol, Belgium
b Biomedical Research Institute, Hasselt University,Diepenbeek, Belgium
c Catholic University Leuven, Leuven, Belgium
d Institut Català de Nanotecnologia, Bellaterra, Spain
e Universitat Autònoma de Barcelona, Bellaterra, Spain
g Vall d Hebron Institute of Research, Barcelona, Spain
h Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain
Sarah Deville
The medical application of gold nanoparticles (GNPs) is promising due to their high biocompatibility, optical and
electrochemical sensing properties, as well as their controlled synthesis, assembly and conjugation with biological
ligands. Many safety studies have focused on pristine GNPs, however, little is known about their behavior in
mixtures with other chemical compounds against a complex biological background. We aimed to understand the
physico-chemical basis for alterations in the safety profile of GNPs when combined with nickel, which is a widely
distributed heavy metal that can form alloys with gold, and has a well documented allergenic and carcinogenic
potential.
To this end, we have focused on allergic sensitization as biological endpoint which was evaluated by means of in
vitro activation of dendritic cells. We performed a detailed study of the physico-chemical interactions between 50-
nm GNPs and nickel(II) sulphate in complex biological matrix using nanoparticle tracking analysis, centrifugal
particle sedimentation, UV-Visible spectroscopy, ICP-MS, ζ-potential determination and proteomics.
While both GNPs and nickel(II) induced an in vitro sensitization response, the cell activation pattern of their
combination was similar to this of nickel(II), suggesting a competitive interaction. Characterization data indicated
that nickel (II) ions did not adsorb onto the GNP surface, but caused a clear shift in the presence and composition of
the GNP-adhered protein corona in biological medium. ICP-MS analyses demonstrated a significant decrease in
GNP uptake by the cells when nickel(II) was present in the environment.
This study highlights the necessity of assessing health risks of nanomaterials in a complex environment reflecting
the real world, and the need to complement such studies with in-depth physico-chemical characterization.
Key words: gold nanoparticles, nickel, mixture toxicology, allergic sensitization, dendritic cells
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Monocytic myeloid-derived suppressive cells as a new component of the carcinogenic response to carbon
nanotubes
Virginie d’Ursel de Bousies1, Marie-Astrid Parent1, Micaela Orsi1, Francine Uwambayinema1, Raynal Devosse1,
Yousof Yakoub1, Nadtha Panin1, Mihaly Palmai-Pallag1, Pierre van der Bruggen2, Christian Bailly3, Riccardo
Marega4, Dominique Lison1, François Huaux1.
1Louvain centre for Toxicology and Applied Pharmacology (LTAP), Institut de Recherche Experimentale et Clinique
(IREC), Université catholique de Louvain, Brussels, Belgium. 2Ludwig Institute for Cancer Research, Brussels Branch,
de Duve Institute, Université Catholique de Louvain, Brussels, Belgium. 3Bio and Soft Matter (BSMA), Institute of
Condensed Matter and Nanosciences (IMCN), Université catholique de Louvain, Louvain-la-Neuve, Belgium.
4Departement of Chemistry, Université de Namur, Namur, Belgium.
The tumor microenvironment of fibre-induced malignant mesothelioma fosters immunosuppressive responses to
counteract effective T lymphocyte surveillance and permit tumor evasion. In this study, we provide evidence that
the initial response to carcinogenic multi-walled carbon nanotubes (CNT-7) comprise an immunosuppressive
component characterized by a specific accumulation of monocytic Myeloid Derived Suppressor Cells (M-MDSC). We
observed that long and short mesotheliomagenic CNT-7 injected in the peritoneal cavity of rats induced, like
asbestos, an early and selective accumulation of monocytic cells (CD11b/cint and His48hi) which possess the ability
to suppress polyclonal activation of T lymphocytes in vitro. These M-MDSC persisted during the development of
peritoneal mesothelioma in CNT-7-treated rats but were only transiently recruited after non-carcinogenic CNT
(CNT-M) or silica injection. Peritoneal M-MDSC were not accumulated in mice resistant to mesothelioma develo
pment upon CNT-7. Our data provide new insight on how carcinogenic CNT may establish an early
immunosuppressive microenvironment facilitating mesothelioma development. The remarkable specificity of the
M-MDSC accumulation after carcinogenic CNT exposure demonstrates the interest of this response for detecting
the ability of new nanomaterials to cause cancer.
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Early and sustained immunosuppressive macrophage response to carcinogenic carbon nanotubes in a rat
mesothelioma bioassay
Marie-Astrid Parent(1), Virginie d’Ursel de Bousies(1), Micaela Orsi(1), Francine Uwambayinema(1), Raynal
Devosse(1), Yousof Yakoub(1), Nadtha Panin(1), Mihaly Palmai-Pallag(1), Pierre van der Bruggen(2), Christian
Bailly(3), Riccardo Marega(4), Dominique Lison(1), François Huaux(1).
(1)Louvain centre for Toxicology and Applied Pharmacology (LTAP), Institut de Recherche Experimentale et Clinique
(IREC), Université catholique de Louvain, Brussels, Belgium. (2)Ludwig Institute for Cancer Research, Brussels
Branch, de Duve Institute, Université Catholique de Louvain, Brussels, Belgium. (3)Bio and Soft Matter (BSMA),
Institute of Condensed Matter and Nanosciences (IMCN), Université catholique de Louvain, Louvain-la-Neuve,
Belgium. (4)Departement of Chemistry, Université de Namur, Namur, Belgium.
Marie-Astrid Parent (Master student)
The asbestos-like toxicity of engineered carbon nanotubes (CNT), notably their capacity to induce malignant
mesothelioma (MM), is a serious cause of concern for public health. MM is a rare cancer affecting the serous
membrane of pleural and peritoneal cavities, highly refractory to therapy. Intensive research efforts are therefore
needed to better understand the pathomechanisms of CNT-induced MM. The present project deals with
immunosuppression during the mesothelial response to CNT. There is growing evidence that tumors harbor
immunosuppressive cells that inhibit both innate and adaptive immunity, subverting immune surveillance and
preventing efficient natural or therapeutic anti-tumor immune responses. We aim to determine if the carcinogenic
response to Mitsui-7 CNT (CNT-7) is associated with the accumulation of immunosuppressive macrophages. We
used a Wistar rat peritoneum model which allows directly exposing mesothelial cells to CNT, and easily sampling
the mesothelia l cavity for monitoring macrophage responses during the carcinogenic process. We show that FACS-
sorted eosinophilic CD11b/chi and His48int macrophages present in CNT-7-induced mesothelioma
microenvironment suppress polyclonal activation of T lymphocytes in vitro. These inhibitory macrophages are
already present during the early response to carcinogenic CNT (day 1 to 30), well before the establishment of
mesothelioma. Immunosuppressive peritoneal macrophages were not observed in mice, which are resistant to
mesothelioma development upon CNT-7. RTqPCR revealed that peritoneal macrophages purified from CNT-treated
rats (day 1) highly expressed the immunosuppressive mediators IL-10 and Arginase-1 in comparison to naive
peritoneal macrophages or macrophages obtained after silica, a particle that does not induce mesothelioma.
Altogether, our data demonstrate that carcinogenic CNT possess the intrinsic capacity to induce a preferential,
rapid and sustained accumulation of immunosup pressive macrophages before mesothelioma is established. These
data provide new insight into the possible contribution of immunosuppression in the early pathogenic processes of
CNT-induced mesothelioma.
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Effects of genotoxicity, DNA methylation and transcriptional changes by Carbon Nanotube exposure in vitro
Deniz Öner(1), Mathieu Moisse(2), Bram Boeckx(2), Manosij Gosh(1), Eveline Putzeys(1), Katrien Poels(1), Radu
Cornelieu(1), Katrien Luyts(1), Ali Tabish(1), Lode Godderis(1), Diether Lambrechts(2), Peter Hoet(1)
(1)Department of Environment and Health, Laboratory of Pneumology, KU Leuven, Leuven, Belgium
(2) Department of Oncology, Laboratory of Translational Genetics (Vesalius Research Center), KU Leuven, Leuven,
Belgium
Deniz Öner
Production and use of carbon nanotubes (CNTs) augmented the last decade, increasing the risk of exposure. Cyto-
genotoxicity of CNT are studied previously, however, it is not known whether CNT exposure induce alterations in
epigenomics and transcriptomics of the pulmonary cells.
We investigated cyto-genotoxicity, epigenetic and transcriptional effects induced by multi-walled-CNTs (MWCNTs)
and single-walled-CNTs (SWCNTs). Two doses (25-100 µg/ml) were evaluated by means of the FpG Comet assay and
the micronucleus assay (with and without incubation of CytochalasinB) in 16HBE (bronchial epithelial) and THP1
(monocytic) cell lines, exposed for 24h. We further, analysed the global DNA-methylation & hydroxymethylation
measured by LC-MS/MS and gene-specific alterations measured by Infinium HumanMethylation450 BeadChip
Array. Next, based on our gene clustering analysis, we analysed and confirmed significantly differentially
methylated genes for their expression to RNA by using RNA-seq.
CNTs induced significant tail DNA and tail moment in both cells types after 24h exposure compared to controls (up
to 10 fold). Moreover, oxidative DNA damage was observed. Frequency of micronucleus was increased when
16HBE cells were exposed to SWCNTs and MWCNTs (1.5 fold). No significant increase in micronucleated cells in
THP1 cells were observed after 24h. CBPI (CytochalasinB block proliferation index) was not significantly altered with
CytB incubation in tested conditions. However, cell cycle phases were significantly disturbed in 16HBE cells.
Although, global DNA-methylation was not significantly altered after 24h exposure, gene (and cell) specific
alterations were observed. Consequently our RNA data confirm 33 genes out of 76 that are significantly
differentially methylated and expressed by MWCNT and/or SWCNT exposure. Based on our system-based KEGG
(Kyoto Encyclopedia of Gene and Genomes) pathways and GO (Gene Ontology) annotation analysis, we identified
pathways and g enes that are in the oncogenic, cell cycle, apoptosis, translational, cell survival pathways by means
of DNA methylation and/or RNA transcription.
Our results demonstrate acute epigenetic and subsequent transcriptional alterations in the pulmonary important
cells which may indicate disease progression after CNT exposure. Furthermore, we propose potential activation of
important pathways and biomarkers in the epithelial lung tissue after CNT exposure.
Grant: Stichting tegen Kanker (Agreement no:2012-218, Project no: Project N° 3M150270)
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The Belgian Society for Toxicology and Ecotoxicology
ABSTRACTS Posters
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Counting asbestos bodies in bronchoalveolar lavage: a time-trend analysis and systematic review
Valerie Nuyts: Centre for Environment and Health, Department of Public Health and Primary Care, KU
Leuven, Belgium
Hadewijch Vanhooren: Centre for Environment and Health, Department of Public Health and Primary
Care, KU Leuven, Belgium
Kristiaan Nackaerts: Department of Respiratory Diseases, University Hospitals Leuven, University of
Leuven, Leuven, Belgium
Benoit Nemery: Centre for Environment and Health, Department of Public Health and Primary Care, KU
Leuven, Belgium
Valerie Nuyts
Introduction
Asbestos bodies (AB) in Bronchoalveolar lavage (BAL) could be quantified by light microscopy: a
concentration higher than 1 AB.ml-1 demonstrates an asbestos exposure higher than the overall
population.
Aims and objectives
The first aim was to assess clinical and exposure characteristics, as well as possible time trends, among
patients in whom AB had been measured in BAL.
We also did a systematic review of the literature on this topic.
Methods
BAL was available from 578 subjects over a period from January 1997 until December 2014, samples
were obtained by bronchoscopy. The processing of samples and the microscopic analysis were done by a
single expert and 76% of samples came from a single tertiary care hospital, allowing clinical and
exposure data to be extracted from patient files.
For the systematic review, databases (Medline and Embase) were searched for relevant articles on the
subject, and 62 articles were selected for a comprehensive analysis.
Results
The study population had a mean age of 62.5 (±12.4) years and 95% of the population was male. The
concentration was higher than 1 AB.ml-1 in 39.4% of the cases and exceeded 5 AB.ml-1 in 17.8%. A
significant decrease in AB concentrations was apparent over the years. High AB concentrations generally
corresponded with occupations with (reported) high asbestos exposure. AB concentrations were higher
among patients with asbestosis and pleural plaques, when compared to other disease groups.
Nevertheless, a substantial proportion of subjects with likely exposure to asbestos did not exhibit high
AB counts.
Results from the systematic review were comparable with our own retrospective study; subjects with
asbestos exposure are more likely to have high asbestos body concentrations. This is also the case for
patients with asbestos-related diseases, especially for patients with ILD (asbestosis included).
Conclusions
This retrospective study of a large clinical population and systematic review supports the value of
counting AB in BAL as a complementary approach to assess past exposure to asbestos.
Keywords: asbestos bodies, brochoalveolar lavage, systematic review, retrospective study
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Are lithium-containing materials used in batteries representing a respiratory hazard?
Violaine Sironval (Louvain centre of Toxicology and Applied Pharmacology, Université catholique de
Louvain, Brussel)
Dominique Lison (Louvain centre of Toxicology and Applied Pharmacology, Université catholique de
Louvain, Brussel)
Sybille van den Brule (Louvain centre of Toxicology and Applied Pharmacology, Université catholique de
Louvain, Brussel)
Violaine Sironval
Extensive research is currently conducted to develop Li-ion batteries for the local storage of energy.
Workers, consumers or general public can be exposed to Li-containing materials used for these
applications, and it is essential to assess their toxicity. Existing knowledge on the toxicity of Li is scarce,
and almost limited to systemic side effects recorded in bipolar patients treated with Li salts. Here, we
evaluated the lung toxicity of 3 leading Li-containing materials (LiFePO4 or LFP, Li4Ti5O12 or LTO, and
LiCoO2 or LCO) and investigated their mechanisms of toxicity.
A size distribution analysis (Anderson cascade impactor) showed that these Li-containing particles
contain a fraction of fine (respirable) particles. At neutral pH (extracellular milieu) particles showed very
different solubilization rates (LFP>LTO>LCO); they were more rapidly solubilized at acidic pH
(phagolysosomal milieu).
C57BL/6 mice were then exposed to Li-particles (0.5-2 mg/mouse) by oropharyngeal instillation. All
materials induced an inflammatory reaction at 18 h or 3 d post-exposure, with a release of inflammatory
cytokines (IL-1β, IL-6 and TNF-α measured by ELISA) already after 18 h. Macrophages and neutrophils
were recruited in the alveoli at 3 d after exposure. LCO was the most cytotoxic (lactate dehydrogenase
release in bronchoalveolar lavage fluid), and displayed the most potent inflammatory activity (cell
recruitment). Two months after administration, an inflammatory reaction involving alveolar
macrophages was maintained only after LCO. LFP, LTO and LCO induced lung collagen accumulation;
fibrotic nodules were observed after LCO only.
Because of the key role of IL-1β in particle-induced inflammation, we analysed the inflammatory
response (3 d) to Li-particles in IL-1β deficient mice. Macrophage and neutrophil recruitment, as well as
IL-6 secretion, were significantly reduced after LCO administration, while IL-1β deficiency did not affect
the response to LFP and LTO.
To further understand the mechanism of the inflammatory activity of these particles, we studied their
activity in differentiated THP-1 macrophages in vitro. LFP, LTO and LCO induced the secretion of IL-1β
that was inhibited by Cytochalasin D (phagocytosis inhibitor) and CA-074 (cathepsin B inhibitor). Particle
uptake and inflammasome activation contribute, therefore, to IL-1β secretion induced by Li-containing
particles. These results also suggest that the inflammatory response is not driven by ions released
extracellularly.
We conclude that Li-containing materials may represent a respiratory hazard. The role of intracellular
solubilization and, specifically, the implication of Li and Co ions in the inflammatory response to LCO are
currently investigated.
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Chronic mouse model of chemical-induced asthma
Lore Pollaris1, Fien Devos1, Peter H.M. Hoet1, Benoit Nemery1 and Jeroen A.J. Vanoirbeek1
1Centre for Environment and Health, Department of Public Health and Primary Care,
Lore Pollaris
Toluene 2,4-toluene diisocyanate (TDI) is one of the basic materials for the production of polyurethanes and is a
well-recognized cause of occupational asthma. In an acute mouse model of TDI we were able to induce several key
hallmarks of occupational asthma, namely an early ventilatory response after intranasal challenge, airway
hyperreactivity (AHR), and airway inflammation (Vanoirbeek et al, 2004). Yet, in this acute model, no airway
remodeling is present. Therefore, we developed a chronic mouse model of chemical-induced asthma.
On days 1 and 8, BALB/c mice were dermally treated (20µl/ear) with 0.5% TDI or the vehicle AOO (3:2). Starting
from day 15, they received under light anesthesia 5 times in a week, for 5 weeks long, intranasal challenges with
0.1% TDI or AOO (3:2). Once in a week, the early ventilatory response was measured using the double chamber
plethysmograph (DCP). One day after the last challenge, airway hyperreactivity (AHR) to methacholine was
assessed, followed by an evaluation of pulmonary inflammation in bronchoalveolar lavage (BAL). As immunological
parameters, lymphocyte subpopulations (CD4+, CD8+, CD25+ and CD19+) and the cytokine production profile in
auricular and cervical lymph nodes were measured. Blood was sampled to determine total serum IgE.
The early ventilatory response of the airways showed that there was a decrease of breathing frequency during the
TDI challenges. After the last challenge, airway hyperreactivity, indicated by increased airway resistance and tissue
elastance was present in TDI challenged mice. Yet, no airway inflammation could be found in the BAL of these mice.
Mice dermally treated with 0.5% TDI were sensitized indicated by a proliferation of both T- and B-cells in the
auricular and cervical lymph nodes. The cytokine production profile of the draining lymph nodes and serum IgE
levels still need to be analyzed
The mice exposed to TDI were sensitized, resulting in airway hyperreactivity, yet airway inflammation remained
absent. This could be due to the development of tolerance and down regulation of the lung inflammation (Nials et
al, 2008). Although there was no lung inflammation, irritation of the airways during the chronic challenges with TDI
was indicated by a persistent decrease of breathing frequency, suggesting involvement of neuro-immune
mediators above inflammatory mediators in this process. This mouse model has the important implication that it
can be used to study the disease progress during chronic exposures to TDI.
21
Influence of the gastro-intestinal environment on ingested silver nanoparticles
Laurie Laloux – UCLouvain / Institut des Sciences de la Vie / Louvain-la-Neuve
Yves-Jacques Schneider – UCLouvain / Institut des Sciences de la Vie / Louvain-la-Neuve
Laurie Laloux
1 Study question
Are ingested silver nanoparticles (AgNPs) altered during the digestion process ?
2. Summary answer
AgNPs undergo some modifications due to interactions with the complex environment of the gastro-
intestinal tract.
3. What is known already
Silver nanoparticles (AgNPs) are increasingly employed in the agri-food sector due to their anti-microbial
properties. They are especially included in food supplements, container coatings and packaging.
Therefore, AgNPs could be ingested by consumers and interact with the complex gastro-intestinal
environment before reaching the intestinal mucosa and, if absorbed, the portal and systemic
circulations, and finally tissues. Digestive enzymes, electrolytes and pH variations could alter AgNPs and
consequently influence their biological responses. These modifications must be taken into account to
understand the impact of AgNPs on health and to improve risk assessment.
4. Study design, with materials & methods in brief
In order to investigate the interaction between AgNPs and the gastro-intestinal tract, we developed an
in vitro pre-colonic digestion method for AgNPs < 20 nm (NM-300K, JRC repository, Ispra, IT) divided into
three steps – i.e. salivary, gastric and intestinal – that were simulated by the addition of the major
digestive enzymes and appropriate pH adjustment. At the end of each step, AgNPs were collected and
characterized by various methods: UV-vis spectrophotometry, transmission electron microscopy (TEM),
dynamic light scattering (DLS) and zeta-potential measurement.
5. Main results
The gastric environment induced nanoparticles agglomeration and negatively charged particles became
slightly positive. Both acidic pH and gastric enzymes are involved in these modifications. The intestinal
step allowed the disintegration of clusters and the recovering of dispersed, nanometric silver particles.
However, fully digested AgNPs are more negatively charged than ingested one’s.
6. Wider implications of the findings
The AgNPs features modifications through the in vitro digestion process could impact the following
interaction with cells and tissues, and have to be considered during the risk assessment.
7. Key words: (min 3- max 5 )
Silver nanoparticles; in vitro pre-colonic digestion; TEM; DLS; zeta-potential
22
Silver nanoparticles effects on the NF-B signalling pathway activation
M. Polet; UCLouvain/ISV/BCNT; Louvain-la-Neuve
I. Massart; UCLouvain/ISV/BCNT; Louvain-la-Neuve
Y.-J. Schneider; UCLouvain/ISV/BCNT; Louvain-la-Neuve
Madeleine Polet
1) Abstract title (max 25 words)
Silver nanoparticles effects on the NF-kB signalling pathway activation
2) Name of presenting author
Madeleine Polet
3) Study question
This study aims at evaluating the effect of silver nanoparticles (AgNPs) on the NF-B cascade governing
the inflammation process on an in vitro model of the gut, Caco-2 cells.
4) Summary answer
AgNPs did not show any activation of the signalling cascade while their presence decreases the response
of the cells to an inflammatory cocktail, suggesting anti-inflammatory properties.
5) What is known already
Since ancient times, silver is used for its anti-microbial property. With the advances of nanotechnology,
AgNPs have come on the market with promises of higher efficiency because of their smaller size and
higher reactivity that could, however, raise their toxicity. Their increasing use in the agri-food sector and
hygienic products can lead to ingestion by consumers. Although their cytotoxicity has been largely
studied and proven, AgNPs effects on inflammation are not converging in the literature.
6) Study design, with materials & methods in brief
The cells were incubated with AgNPs (NM-300K from the JRC – 15nm) at non-cytotoxic concentrations
(from 1.5 to 15µg/mL) in the presence or absence of an inflammatory cocktail, developed to activate
this signalling pathway. The effect of AgNPs on the cascade was evaluated at three different levels. First,
the nuclear entrance of p65 was observed on a confocal microscope after an immunostaining and
quantified via an image analysis software. Secondly, Caco-2 cells were transitory transfected with a
plasmid coding for a luciferase under the control of response elements to NF-B. The luciferase amount,
after the incubation, reflects the activation of NF-B in the transfected cells. Finally, the IL-8 chemokine,
target of the signalling pathway, was measured after a 3-hour incubation followed by a washout of 21-
hour.
7) Main results
Results show that AgNPs alone do not activate the NF-B cascade, underlining an absence of pro-
inflammatory properties while they seem to impair the signalling pathway in the presence of an
inflammatory cocktail, suggesting anti-inflammatory properties.
8) Wider implications of findings
This study suggest that even if they are cytotoxic, AgNPs can present anti-inflammatory properties.
9) Key words: (min 3- max 5)
Silver nanoparticles – Inflammation – NF-B signalling cascade - Caco-2 cells
23
Framework to evaluate exposure relevance and data needs for risk assessment of nanomaterials using in vitro
testing strategies
Monita Sharma 1,
Christopher Faßbender 1,
Jo Anne Shatkin 2,
Carolyn Cairns 3,
Richard Canady 4,
Amy J. Clippinger1
1 PETA International Science Consortium Ltd., London, United Kingdom,
2 Vireo Advisors, LLC, Boston, MA, United States,
3 Independent Consultant, Nyack, NY, United States,
4 Neutral Science L3C, Arlington, VA, United States
Christopher Faßbender
Presented here is a multi-stage framework for evaluating the strength of evidence of nanomaterial (NM) exposure
characterization data to optimize the utility of in vitro testing strategies for human health risk assessment.
The initial stage frames the exposure considerations and scenarios of interest in advance of testing to link aspects
such as release points, route of exposure, biological and environmental transformations, dose metrics, and
biological targets in subsequent stages. The second stage considers characterization in the context of a realistic
exposure and the third stage involves designing a testing strategy based on expected exposure conditions. For the
fourth and final stage, a matrix approach is proposed to evaluate the strength of evidence obtained in the first
three stages as a basis for determining the best combination of test conditions and analytical methods available to
characterize and measure exposure based on the NM type.
This framework is intended to aid risk assessors in evaluating the relevance of data from in vitro tests and to
optimize the development of new in vitro testing strategies based on specific exposure scenarios.
Key words: nanomaterials, exposure characterization, human health risk assessment, in vitro testing strategies
24
CON4EI: Consortium for in vitro Eye Irritation testing strategy
An R. Van Rompay1, Els Adriaens2, Nathalie Alépée3, Agnieszka Drzewiecka4, Przemyslaw Fochtman4, Katarzyna
Gruszka4, Robert Guest5, Helena Kandarova6, Joke Lenoir7, Jamin A. Willoughby Sr.8 and Sandra Verstraelen1
1Flemish Institute for Technological Research (VITO NV), Mol, Belgium; 2Adriaens Consulting bvba, Aalter, Belgium;
3L’Oréal Research & Innovation, Aulnay-sous-Bois, France; 4Institute of Industrial Organic Chemistry, Pszczyna,
Poland; Envigo, Derbyshire, United Kingdom; 6MatTek In Vitro Life Science Laboratories, Bratislava, Slovak
Republic; 7Ghent University, Laboratory of Pharmaceutical Sciences, Gent, Belgium; 8Cyprotex US, LLC, Michigan,
USA.
An R. Van Rompay
Assessment of the acute eye irritation potential is part of the international regulatory requirements for testing of
chemicals. All in vitro assays have specific strengths and limitations whether this relates to ranges of irritancy, types
of chemical classes or physical nature of the materials. Therefore, combinations of in vitro assays are needed for
hazard identification and complex safety assessment. Today, these combinations of assays are used by individual
companies as an integral part of their safety assessments, but often with limited scientific knowledge covering their
own specific chemical properties/portfolio needs. This can contribute to a lack of consistency among in vitro test
results within a battery approach or a conflict with available in vivo data. Despite efforts to compare in vitro
methods with in vivo data, the problem is that most of the so far proposed testing strategies for assessment of eye
irritation were not evaluated with the same adequately lar ge set of chemicals.
The main objective of this project (CEFIC LRI-AIMT6-VITO CON4EI) is to develop tiered testing strategies for eye
irritation assessment for all eye irritation drivers of classifications. The irritancy potency of a set of 80 chemicals will
be evaluated. Following eight test methods will be included in this project: BCOP1, ICE1 and STE2, to distinguish
Category 1 vs No Category. EpiOcular EIT2 and SkinEthicTM HCE3 to distinguish Not classified vs classified. To
distinguish between Category 1 and Category 2, the following methods were selected: histopathology in association
with BCOP and ICE, EpiOcular ET-504, SMI, and BCOP-LLBO.
This project will assess the reliability of these in vitro test methods, define applicability domains in terms of ‘drivers
of cla ssification’, strengths and limitations of each method. In this way, we will be able to identify methods that
will fit in a tiered approach to distinguish UN GHS classified Category 1 chemicals versus No Category chemicals and
address the highest industrial gaps namely distinguish between Category 2 versus Category 1 chemicals.
1 regulatory accepted, 2 undergoing regulatory acceptance, 3 mature in development, 4accepted by EPA for testing
of antimicrobial cleaning products.
This research is funded by CEFIC-LRI. We acknowledge Cosmetics Europe for their contribution in chemical
selection.
25
Effect of environmental irritants on the respiratory barrier in a 3D-cell culture model
Sofie Van Den Broucke, Deniz Öner, Hanne Vriens, Peter Hoet.
Centre for Environment and Health, Department of Public Health and Primary Care, KU Leuven
Sofie Van Den Broucke
The human respiratory barrier is an important part of the lung as it serves as a physical fence to limit access of
exogenous compounds to host and plays a role in airway inflammation. Upon inhalation, environmental irritants
under the form of chemicals, dust or fumes can disturb this airway barrier, which might result in increased airway
permeability. In this research we are interested in the effects of environmental irritants on the pulmonary barrier
and how these effects could contribute to the development of airway hypersensitivity, such as irritant-induced
asthma.
To mimic the pulmonary barrier a co-culture model, consisting of cells of the human bronchial epithelial 16HBE14o-
cell line (16HBE) and cells of the human monocytic cell line (THP1) seeded on the apical compartment of a
permeable support, and human lung microvascular endothelial cells (HLMVEC) grown to the basal side, was used.
These co-cultures were exposed to sub-toxic concentrations of graphene, graphene oxide, lipopolysaccharide,
hypochlorite, ammonium persulfate or sodium persulfate. The permeability of the cell layers was evaluated using
measurement of the transepithelial electrical resistance (TEER) and paracellular flux of fluorescent labelled
dextrans. For assessment of the epithelial integrity, immunoflourescent staining of the tight junction (TJ) proteins
ZO-1 and occludin was performed.
Exposure of the co-culture to graphene and graphene oxide resulted in an immediate decrease in TEER compared
to the control. This decrease persisted up to 24 hours after exposure for graphene (256µg/ml and 64µg/ml) and at
high concentrations for graphene oxide (64µg/ml). A concentration of 0.0075% and 0.00375% active chlorine in
hypochlorite lead to a significant decrease in TEER only 24 hours after exposure. The flux of fluorescent labelled
dextrans increased respectively tree- and two-fold after 24 hours of exposure to 256µg/ml and 128µg/ml graphene.
Exposure of 64µg/ml graphene oxide also resulted in a significant increase in flux of fluorescent labelled dextrans.
Preliminary immunofluorescent staining of the co-cultures do not indicate disruption of the TJs.
These results indicate that exposure to the nanoparticles graphene and graphene oxide leads to increased airway
permeability. Further research needs to be performed to investigate how these agents affect the barrier integrity.
By disturbing the pulmonary barrier, access of foreign agents to the supepithelial compartment is facilitated and
epithelial and innate immune cells will get stimulated. This is possibly a mechanism contributing to the
development of airway hypersensitivity, such as irritant-induced asthma.
26
Ag nanoparticles toxicity on Daphnia magna under acute and chronic tests
S. Cambier1, E.J. Keuzenkamp1,2, A. Georgantzopoulou1,3, M. Kruszewski4, A.J. Murk2, A.C. Gutleb1
1 Environmental Research and Innovation (ERIN) Department, LIST, Esch sur Alzette, Grand-duchy of Luxembourg
2 Division of Toxicology, Wageningen University, Tuinlaan 5, 6703 HE Wageningen, The Netherlands
3 Norsk Institutt for vannforskning NIVA, Oslo, Norway
4 Institute of Nuclear Chemistry and Technology, Warsaw, Poland
S. Cambier
Silver nanoparticles are used more and more in consumer products, ranging from textiles, cosmetics, food
packaging to electronics. This increase in their use will finally lead to their release in the environment where they
will inevitably end up in aquatic environments and have a potential impact on their ecosystem. However, the fate
of the Ag NPs in aquatic ecosystems is still not fully understood as well as their impacts on aquatic living organisms.
For this purpose, Daphnia magna, a model species for aquatic environments, were exposed to different citrate
stabilised silver nanoparticles (ci-Ag NPs: 20, 40, and 80 nm), non-citrate stabilised silver nanoparticles (Ag NPs: 20
200 nm), and silver nitrate under acute and chronic exposure conditions to evaluated their potential toxicity. In
parallel to this toxicity assessment the fate of the Ag NPs was investigated by using the NTA approach. In mineral
water the Ag NPs present different behaviour (agglomeration, dissolution etc.) depending on their size and surface
chemistry. Indeed both Ag NPs of 20 nm formed aggregates of 100 nm whereas Ag NPs of 40 and 80 nm were more
stable in exposure media. This study showed that ci-Ag NPs are more toxic in acute conditions than the non-
stabilized. However, under chronic conditions a significant increase in mortality occurred when D. magna was
exposed to Ag NPs of 200 nm at equitoxicity level (EC5 and EC1) compared with the other nanoparticles.
27
Development of a zebrafish embryo test for environmental risk assessment of pharmaceuticals with endocrine
disrupting properties
Ellen D.G. Michiels (1), Lucia Vergauwen (1), Steven J. Van Cruchten (2) and Dries Knapen (1)
(1) Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of
Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium
(2) Applied Veterinary Morphology, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1,
2610 Wilrijk, Belgium
Ellen D.G. Michiels
Pharmaceutical companies are obligated to perform an environmental risk assessment for each new drug that is
launched on the market. The mandatory tests for potential endocrine disrupting compounds require a lot of time
and laboratory animals, which is not consistent with the 3R principle. Therefore, the goal of this study is to develop
a zebrafish embryo test, which is not considered an animal test according to the European regulation on the use of
laboratory animals, and which is capable of detecting and discriminating among 5 endocrine disrupting modes of
action (MOAs). The MOAs that will be studied are estrogen receptor agonism and antagonism, androgen receptor
agonism and antagonism and aromatase inhibition. As a first stage of test development, one pharmaceutical per
MOA is investigated to map differences among the 5 compounds in zebrafish embryos at the morphological,
physiological and molecular level.
So far, we examined the first three compounds on differences at the morphological and physiological level: the
androgen receptor agonist 17β-trenbolone, the androgen receptor antagonist flutamide and the estrogen receptor
agonist 17α-ethinylestradiol (EE2). Embryos were exposed to each of the compounds dissolved in reconstituted
freshwater using 0.01% DMSO as a solvent, starting from 0-2 hours post fertilization (hpf) until 120 hpf. We
observed some similarities as well as some differences in the responses among compounds. Mortality always
occurred in the first 24h after fertilization for all three compounds.
Both receptor agonists showed some sublethal effects, while no sublethal effects were observed after exposure to
the androgen receptor antagonist, flutamide. After exposure to EE2, the embryos showed sublethal, dose-
dependent skeletal malformations of the spine. Cranial malformations were only significant around the LC50
concentration and above. For 17β-trenbolone (androgen receptor agonist) heart rate was already significantly
decreased after exposure to concentrations below the LC5. Growth was decreased below the LC50 concentration
for the androgen receptor agonist, while for the other 2 compounds the decrease was only significant around the
LC50 concentration. Swim bladder inflation and swimming behaviour were impaired for both receptor agonists at
sublethal concentrations.
In future research, effects of endocrine disruption compounds on transcriptional expression of important genes, as
well as the response of transgenic zebrafish lines will be investigated. While the sole use of morphological and
physiological endpoints seems to have limited merit for differentiating among endocrine disruption MOAs, we
expect that the combination of different data types will increase the discriminating potential.
Keywords: zebrafish embryo, endocrine disrupting compounds, pharmaceuticals
28
Development of a zebrafish feeding trial to evaluate food safety.
Isabelle Gabriëls(1), Lucia Vergauwen(1), Marthe De Boevre(2), Sarah De Saeger(2), Ronny Blust(3), Mia
Eeckhout(4), Marc De Loose(5), Dries Knapen(1)
(1)Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp,
Universiteitsplein 1, 2610 Wilrijk, Belgium
(2)Laboratory of food Analysis, Department of Bioanalysis, Ghent University, Ottergemsesteenweg 460, 9000
Ghent, Belgium
(3)Systemic Physiological and Ecotoxicological Reseach (SPHERE), Department of Biology, University of Antwerp,
Groenenborgerlaan 171, 2020 Antwerp, Belgium
(4)Department of Applied Biosciences, Faculty of Bioscience Engineering, Ghent University, Valentin Vaerwyckweg
1, 9000 Ghent, Belgium
(5)Technology and Food Sciences Unit, Institute for Agricultural and Fisheries Research (ILVO), Burg. Van
Gansberghelaan 115, 9820 Merelbeke, Belgium
Key words: Food safety - Feeding trials - Zebrafish
The safety of food components, additives and contaminants is generally evaluated using rat feeding trials to assess
(sub)chronic toxicity and reprotoxicity. The development and implementation of an alternative zebrafish feeding
trial would refine these testing strategies by reducing the cost, replacing the use of mammals by a lower vertebrate
species, and by facilitating reproductive and multigenerational studies. When testing food components (e.g.
GMOs), a substantial part of the food is substituted by the component under evaluation, possibly interfering with
nutritional requirements which has led to severe criticisms on rat feeding trials. Therefore, when developing a
zebrafish feeding trial to investigate food components, the extent of component substitution still processable by
the zebrafish metabolism (maximum tolerable percentage) should be assessed prior to the component evaluation
trial.
Since we will conduct a feeding trial with GM maize in a later phase of our study, we defined the maximum
tolerable percentage of maize. First, we formulated an experimental fish feed based on the composition of
commercial fish feeds. The maize was introduced in the feed by a stepwise substitution of a wheat component
(25% of the whole feed). In this way, 6 experimental feeds were formulated ranging from 0% to 25% of maize in
steps of 5%. We compared these experimental feeds to three different commercial feeds in a one month zebrafish
feeding trial. We investigated the following endpoints: relative condition factor and growth, hepatosomatic and
gonadosomatic index (HSI and GSI respectively), reproduction, energy budget and feed digestibility. The growth of
fish fed with either 25% wheat or 25% maize slightly decreased, possibly indicating that a combination of wheat
and maize in the diet results in a more balanced carbohydrate composition for zebrafish. Next, we observed an i
ncreased HSI in males when the percentage of maize in the feed increased. The HSI was significantly increased
when males were fed either 20% or 25% maize substitution compared to the control feeds. Measurement of feed
digestibility showed a clear decrease in carbohydrate uptake when fish were fed with an increasing percentage of
maize substitution, and this decrease was significant from a 15% maize substitution onward.
Based on these results, we selected an adequate percentage of 15% of maize substitution for conducting the
feeding trial with GM maize. This selection was based on two criteria: (1) the biological limits of the maize
component in the feed of zebrafish should be respected, (2) the amount of maize substitution should allow us to
observe effects of GM maize if there are any. We suggest that our approach of first determining maximum
tolerable food component substitution rates before carrying out feeding trials could be a valuable addition to
existing protocols.
29
Development of an adverse outcome pathway for “thyroperoxidase and/or deiodinase inhibition leading to
impaired swim bladder inflation”
E. Stinckens (1), L. Vergauwen (1), AL. Schroeder (2,6), W. Maho (3), BR. Blackwell (2), H. Witters (4), R. Blust (5),
GT. Ankley (2), A. Covaci (3), DL. Villeneuve (2), and D. Knapen (1)
(1) Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium (2) US EPA, ORD NHEERL, Mid-Continent Ecology Division, 6201 Congdon Dlvd, Duluth, MN 55804 USA (3) Toxicological Centre, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium (4) Applied Bio & molecular Systems (ABS), Flemish Institute for Technological Research (VITO), Boeretang 200, 2400 Mol, Belgium (5) Systemic Physiological and Ecotoxicological Research (SPHERE), Department of Biology, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium (6) University of Minnesota – Twin Cities, Water Resources Center, 1985 Lower Buford Circle, St. Paul, Minnesota, 55108, USA
The vast number of industrially produced chemicals has – in the light of the 3R principle – generated a strong focus
on alternative test development for ecological risk assessment. Therefore, we are developing a non-animal testing
strategy for the prediction of chronic aquatic toxicity including in vitro tests and in vivo ZFET (zebrafish embryo
acute toxicity test, OECD TG 236) assays. Moreover, we are assessing the feasibility of using an adverse outcome
pathway (AOP)-based approach for this purpose.
Thyroid hormone (TH) disruption is increasingly recognized as an important mode of action and a major regulatory
concern as well. However, endocrine properties of chemicals are often not well-known. Therefore, we are
developing an AOP related to TH synthesis and activation, encompassing thyroperoxidase (TPO) and/or deiodinase
(DIO) inhibition, impacting swim bladder development and inflation. Impaired swim bladder inflation is considered
an ecologically relevant adverse outcome as it affects endpoints including feeding behaviour and predator
avoidance, resulting in lower survival probability.
A hypothesized AOP was developed over several iterations using refined 120 hours post fertilization (hpf) ZFET
tests, exposing zebrafish embryos to mercaptobenzothiazole (MBT), methimazole (MMI), propylthiouracil (PTU),
and iopanoic acid (IOP). Results show that exposure to MBT and MMI, two TPO inhibitors, do not directly impair
posterior chamber inflation at 120 hpf, while IOP, a DIO inhibitor, and PTU, which inhibits TPO and DIO, do. Since
the posterior chamber inflates during early development, we hypothesized that embryonic TPO activity is not
essential for posterior chamber inflation because of the presence of maternal T4 (prohormone), while deiodinase
activity is needed to activate T4 into the biologically active T3. Furthermore, we hypothesized that both TPO and
DIO are needed to synthesize and activate THs at later developmental stages, since maternally derived THs are
depleted and cannot offset TPO inhibition. Subsequently, both TPO and DIO inhibitors were predict ed to impair
anterior chamber inflation, which occurs during late development.
To investigate this hypothesis, a fish early-life stage toxicity test (FELS, OECD TG 210) was performed, exposing
zebrafish embryos to MBT until 32 days post fertilization (dpf). MBT exposure resulted in impaired anterior
chamber inflation at 21 dpf, in line with the hypothesized AOP. Furthermore, a clear relationship between T4 levels
and anterior chamber surface was found, suggesting that anterior chamber inflation is influenced by THs.
Finally, we investigated the potential of several compounds to inhibit TPO and/or DIO at the enzyme level. The
results will be used to (1) investigate a correlation between the TPO/DIO inhibitory potential of compounds and
acute and chronic data, (2) build a model to predict swim bladder inflation and (3) validate the impact on swim
bladder inflation using ZFET and FELS tests.
Keywords: Adverse outcome pathway; thyroid hormone; swim bladder inflation; zebrafish embryo
30
An innovative in vitro real time monitoring method to evaluate the potential toxicology of nanomaterials
Jacques-Aurélien Sergent, Toxicological and Environmental Risk Assessment Unit, Solvay
Erik Van Miert, Toxicological and Environmental Risk Assessment Unit, Solvay
Due to the growing demand for toxicological profiling of nanomaterials, for instance in support of grouping
approaches and R&D projects, there is a need for innovative in vitro screening methods capable of handling a wide
variety of (nano-)materials and providing robust results. In vitro assays are partial “models” of the in vivo or clinical
reality and in particular nanomaterials have shown the capacity to interfere with the methodological principles of
these models leading to artefacts of little toxicological relevance. The in vitro XCELLigence platform (ACEA
Biosciences) based on the principle of impedancemetry was identified as an interesting candidate. Its measurement
principle is relatively indifferent to the physico-chemical characteristics of the substance tested and it allows the
real-time monitoring of cell homeostasis, adhesion and proliferation of adherent cells. The assay enables the
detection of effects like cytostasis, cytotoxicity, receptor-mediated si gnalling or modifications of cell-cell
interactions
As a test case, we investigated the effects of expanded graphite, carbon black, graphene oxide, reduced graphene
oxide on HuH7 cells. The specific surface of these uncoated and non-soluble materials ranged between 31 and 620
m²/g. The proliferation and cytoxicity were investigated at 5000 cells/well over a 125h time period whereas cell-cell
interaction was investigated at confluence (30000 cells/plate) over an exposure time of 24h. A negative control and
five dose levels (up to 100 µg/ml) per material were tested. No technical issues due to the materials tested were
encountered and all nanomaterials yielded interpretable results.
On a surface-basis, the biological activity showed the following increasing trend 1) carbon black 2) reduced
graphene oxide 3) graphene oxide and 4) expanded graphite. Overall, these results are in agreement with available
literature.
The in vitro platform XCELLigence confirmed its potential as a useful and rapid first line screening tool for
nanomaterials and it will be implemented as such in Solvay. The main strengths of this screening tool are its
flexibility towards the substances which can be tested and the types of adherent cell lines which can be used, and
the fact that both short term (transient) and long term effects on cellular kinetics can be studied. The possibility to
combine this assay with confocal microscopy or flow cytometry is an additional plus. In a future where more data
will be available and analysed, mode-of-action specific alerts might be deduced from the dose- and time-specific
characteristics of the impedancemetry readouts.
31
EcoTexNano: Nanomaterials offer interesting finishing opportunities to textiles, but what about the risks of
nanomaterials to human health and the environment?
Erik Wuyts, CENTEXBEL
Raquel Villalba - LEITAT
Vincent Jamier - LEITAT
Nanomaterials can contribute great functionalities to textiles such as a microbial property, UV-protection, soil-release and flame retardency. However, inclarity remains concerning the risks to human health and the environment. The aim of EcoTexNano is to demonstrate and improve the environmental performance of textile finishing processes incorporating nanoparticles. Pilot scale trials are performed to provide evidence of best practice in the application of nanomaterials compared to conventional finishing chemicals. Based on data obtained from these pilot scale trials, environmental, health and safety impacts are assessed for both "Nano - finishes" and "conventional textile finishes."
The main objectives of the project are to:
1) Improve the added value of textiles by providing interesting properties through the application of nanomaterials.
2) Identify and reduce the environmental and health impacts by carrying out a comprehensive Life Cycle Assessment and Risk Assessment/Risk Management of the selected nanomaterials.
3) Contribute to the regulatory work initiated at the European level on nanomaterials
4) Increase the consumer’s awareness about EHS- issues related to the implementation of nanomaterials in the textile sector
The final project results will be assembled in a user-friendly, WEB-BASED TOOL to support future design and implementation of textile finishing processes for nanomaterials.