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SEACSAFETY & ENVIRONMENTAL ASSURANCE CENTRE
Overcoming barriers to non-animal risk assessments for anti-androgenic effects in humansMatthew Dent1, Marguerite Vantangoli Policelli2, Chloe Bars2, Paul Carmichael1, Kim Boekelheide 2, Francis Martin3
Date: 06/09/2016
SAFETY SCIENCE IN THE 21ST CENTURYFor more information visit www.tt21c.org
ABSTRACT
Toxicology testing is undergoing a transformation from a system based on high-dose studies in animals to one founded primarily on in vitro methods that evaluate changes in normal cellular signalling pathways using human-
relevant cells or tissues. This is a challenge for anti-androgenic effects in humans, since some parts of the hypothalamus-pituitary-testicular (HPT) axis are not well represented by accepted in vitro methods. These include
key events relating to gonadotropin releasing hormone (GnRH) signalling which could be affected at the level of the hypothalamus and pituitary. In vitro tools are needed to characterize either specific effects (such as GnRH
receptor antagonism) or non-specific effects (such as general toxicity causing a reduction in gonadotropin release) before an integrated model of the HPT axis can be described. We have been evaluating how this could be
achieved using human non-pituitary cells that express GnRH receptors and synthesize gonadotropins. Furthermore, tools to characterize a tipping point between endocrine activity and adversity need to be developed to
allow an assessment that is more representative of the underlying biological response to endocrine active chemicals. To this end we have been developing and characterizing human derived scaffold-free prostate
microtissues to provide morphological and molecular readouts to identify exposures that lead to adverse responses. Our ambition is to use these tools in an exposure-led safety assessment to enable robust safety decision
making for endocrine active chemicals without use of animals.
1Unilever Safety and Environmental Assurance Centre, Colworth Science Park, Bedfordshire, UK2Department of Pathology and Laboratory Medicine, Brown University, Providence, RI, USA3Lancaster Environment Centre, University of Lancaster, Lancaster, UK
Liu et al 2011
http://dx.doi.org/10.1177/1947601911409744
BACKGROUND
CONCLUSIONS
Progress is being made in filling some of the gaps in the tools needed for non-animal safety assessments for anti-androgenic effects. For example, non-
pituitary human neuronal cell lines may provide a useful screen for perturbations in GnRH signalling, and co-culture of human prostate epithelial and stromal
cells in scaffold-free hydrogels are showing promise as a means to develop more in vivo like cell models. For these to be useful in decision making they
need to be used as part of an exposure-led safety assessment approach.
Fig. 1: Overview of the HPT axis. Disturbance
in signalling pathways anywhere in the axis
could ultimately result in adverse effects
depending on the site of action, potency of the
toxicant, level and timing of exposure. In vitro
tests for (anti-)androgenicity have tended to
focus on androgen receptor (AR) agonism and
antagonism. However chemicals may cause
adverse effects anywhere in the HPT axis via
either specific (receptor mediated) or non-
specific modes of action.
GnRH: gonadotropin releasing hormone; LH:
Luteinizing hormone; FSH: Follicle stimulating
hormone; T: Testosterone; DHT:
Dihydrotestosterone; 5α-R: 5 alpha-reductase
Dent et al. (2015) Env. Int. 83: 94-106
Wilson et al. (2006) J. Endocrinol. 191: 651-663
Rosati et al. (2011) J. Steroid Biochem. Mol. Biol. 124: 77-83
Ozone et al. (2016) Nature Communications 7:10351
Suga et al. (2011) Nature 480: 57-64
Cell seeding density alters cell sorting
Consumer use and internal exposure assessment supported by PBPK models
Develop high content in vitro assays in human cells and models to interrogate pathways of concern
Evaluate the dose-response from chosen assays
Computational models of the circuitry of relevant pathways
Risk assessment based on exposure levels below significant pathway perturbations
Fig 2: Key to developing a non-animal risk
assessment for anti-androgenic effects in
humans is the use of an exposure-led
approach using some (or all) of these
components. Success is dependent on the
availability of a broad suite of in vitro tests that
cover modes of action that could lead to
perturbations in androgen signalling (box 2),
and the ability to distinguish between an
adaptive response (which may be indicative of
endocrine activity) and an adverse response.
Our work seeks to address these two areas.
PBPK: physiologically based pharmacokinetic
modelling.
Key Gap 1: in vitro tools covering events across the HPT axis
Although rodent gonadotrope cell lines exist, there are no commercially-available human cell lines
used for evaluating perturbations in GnRH signalling. Models derived from stem cells are in
development (Suga et al. 2011; Ozone et al. 2016) but are not yet commercially available. We
therefore evaluated whether human non-pituitary neuronal cell lines could provide a surrogate
screen for the ability of a chemical to perturb GnRH signalling. For a cell system to be useful in
characterizing the effects of chemicals on GnRH-mediated release of gonadotropins, it needs to
express GnRH Type 1 receptors and respond to GnRH signals by increasing production of
gonadotropins using the same signalling pathways as human gonadotrope cells. For example, SH-
SY5Y neuroblastoma cells express GnRH Type 1 receptors and reportedly respond to GnRH
stimulation (Wilson et al. 2006). We performed RT-PCR on these cells and confirmed that although
they do not express mRNA for FSHβ, they respond to GnRH stimulation by increasing both GnRHR
and LHβ gene expression (Figure 3). The observation that these cells do not express mRNA for
FSHβ is clearly a limitation of this cell type. However, if the signalling pathways present in this cell
line are analogous to those present in normal human gonadotrope cells this cell line could still be a
useful tool in determining the likelihood that chemical exposure could perturb GnRH signalling.
Fig 3: SH-SY5Y cells from The European
Collection of Authenticated Cell Cultures (ECACC)
were cultured in a monolayer in Ham's F12:Eagle’s
Minimal Essential Medium (EMEM) (1:1)
supplemented with final concentrations of 2mM
Glutamine, 1% Non Essential Amino Acids
(NEAA), 15% Heat Inactivated Fetal Bovine Serum
(FBS) and 100U/ml/100µg/ml
Penicillin/Streptomycin, in an atmosphere of 5%
CO2 in air at 37ºC for 48-hours, then placed in
serum-free medium (Dulbecco’s Modified Eagle’s
Medium with 1% insulin/transferrin/sodium selenite
supplement) for 22.5 hours. Cultures were then
supplemented with GnRH solution at 0 (control),
0.1, 1 or 10 nM for 90-minutes, as exposures in
this range were expected to activate the GnRHR
(Wilson et al. 2006, Rosati et al. 2011). At 90-
minutes RNA was extracted and RT-PCR was
used to detect and changes in the expression of
GnRHR, LHβ, or FSHβ using TaqMan® gene
expression assays. RQ = relative quantification (to
0 nM)
Fig 4: SH-SY5Y cells are known to differentiate
and differentially express genes dependent on their
culture conditions. As part of our characterization
we were interested to see if the expression of
these target genes changed over time, when the
cells were cultured in the complete medium
described above for different lengths of time. We
demonstrated that at 72-hours after seeding there
remained no FSHβ gene expression, and LH β
gene expression was unchanged. However
GnRHR was markedly increased between 24- and
72-hours after seeding. We also detected changes
in the proportion of cells in different phases of the
cell cycle over this timeframe, indicating a greater
proportion of cells in G0 or G1 at 72-hours
compared with 24-hours (data not shown). RQ =
relative quantification (to 24-h)
Fig 4: RT-PCR analysis of cells
collected at different timepoints after
seeding (24- or 72-hours).
Endogenous control β-actin.
Key gap 2: in vitro tools more representative of in vivo biology
Chemicals that are endocrine active are not necessarily endocrine disrupters. Whether a chemical
with endocrine activity in vitro will cause an adverse response in vivo depends on many factors,
including the properties of the chemical such as its potency, the level of human exposure, the timing
of exposure (taking into account critical windows of development) and the duration of exposure
(short term or chronic). The majority of in vitro tests for endocrine activity rely on detecting a
biological response (e.g. transcriptional activation) that may or may not lead to an adverse response
in vivo. Those assays that provide a functional response to endocrine active chemicals tend to rely
on 2D cultures using cell systems that are not necessarily representative of the biology of normal
endocrine sensitive tissues in vivo. We envisage that a 3D system will be critical in determining
whether in vitro endocrine activity (e.g. androgen receptor antagonism detected in a reporter gene
assay) is likely to result in an adverse response in vivo at relevant exposures. We have therefore
been evaluating whether prostate cells (RWPE-1 epithelial cells and WPMY-1 myofibroblast cells)
can be co-cultured in a 3D scaffold-free system to provide a more in vivo-like phenotype, enabling us
to identify morphological and molecular biomarkers to differentiate between endocrine activity and
adversity.
00.
03 0.1
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WPMY-1 Testosterone Cell Count
nM Testoterone
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WPMY-1 DHT Cell Count
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WPMY-1 Estradiol Cell Count
nM Estradiol
Cell N
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* **
******** ****
****
C D
nM T
Fig 5: Prostate cell lines are responsive to
androgen when cultured as two-dimensional (2D)
monolayers. When imaged using the Opera Phenix
high-content confocal, RWPE-1 epithelial cells
grown as 2D monolayers exhibit typical
cobblestone morphology (A), while WPMY-1
myofibroblast cells have spindle-like morphology
(B). RWPE-1 cells exhibit a concentration-
dependent increase in proliferation following
exposure to testosterone (T) for 24 hours (C), while
WPMY-1 cells are responsive at concentrations of
0.3 and 1nM (D). Immunfoluorescence - blue:
Hoechst 33342, yellow: rhodamine phalloidin, *
p<0.05, **** p<0.0001
A B
24 hours 48 hours 72 hours
1:1
01
:14
:1
A B CA
D E F
G H I
Fig 6: Cell seeding density alters cell sorting and
structure in 3D RWPE-1/WPMY-1 co-cultures.
An initial seeding ratio of 1 RWPE-1
(CellTracker Red) : 10 WPMY-1 stromal
(CellTracker Green) cells results in a center core
of RWPE-1 cells surrounded by and exterior of
WPMY-1 cells at 24 (A), 48 (B) and 72 hours
(C). A 1:1 ratio of RWPE-1 and WPMY-1 cells
results in a bi-lobulate microtissue (D-F). A 4:1
ratio of RWPE-1 to WPMY-1 cells drives the
formation of a less condensed, disorganized
microtissue (G-I). Images were obtained using
an Opera Phenix high-content confocal as z-
stacks with z distance of 5μm, and images are
presented as maximum projections. Current and
future work is focused on assessing the
response of these microtissues to androgens
and anti-androgens, and assessing their protein
and gene expression.