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.

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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.