Application of a novel, pathway-based screen for Developmental ...
Post on 18-Jan-2017
214 Views
Preview:
Transcript
Application of a novel, pathway-based screen for Developmental And Reproductive Toxicity (DART)
Dr. Marjolein WildwaterProgram leader alternative test systems HAN BioCentre
Dr. Chantal SmuldersShell
Alternative in vivo 3R species for developmentaland reproduction (DART) hazard prediction
Unique features • Well known 3D systems
with tissues and organs
• Conserved molecular andcellular pathways
• Huge amount of availabletools/technologies
• High throughput
• Low cost
• Rapid generation time
• 3R
Challenges
• Not human
• Further requirement forvalidation and determinationof applicability domain
• Not yet accepted byregulators
A combinatorial approach to identify biomarkers and AOPs for developmental and reproductive
toxicity
D. discoideum, C. elegans and D. rerio as alternative test systems for developmental and
reproductive toxicity
You tube: by Tglab You tube: tony hyman lab You tube: Karlstrom and Kane
What makes these organisms predictable for effects in humans???
Shell International B.V. April 2016
C. elegans genome wide RNAi library
Easy AOP identification High throughput methodology
D. rerio robotic injection equipment
Craniofacial bone reporter Notch receptor
C. elegans
Fluorescent reporters
Available tools allow rapid and unique testing
Comparable organs, tissues and cell biology; conserved genetic responses/pathways!
http://www.sfu.ca/biology/faculty/hutter/hutterlab/research/Ce_nervous_system.html
• Muscles• Skin• Neurons• intestine C. elegans neurons visualised by GFP
D. rerio• Muscles• Skin• Neurons• Intestine• Heart• Eye• cartilage
C. elegans cell lineage is known and invariable
An example: Pax6, an evolutionary conservedgene for eye development
Washington et al PLoSBiol e1000247
Pax6 important in C. elegans for normal headdevelopment
Cinar and Chisholm., Genetics 168: 1307–1322
Ultimate goal: medium throughput tiered 3R-approach to predict DART potential in man
Phenotypic responses of 3 species to selected chemicals
Internal dose assessment exposure
Mapping of adverse outcome pathways by RNA-sequencing, RNAi and kinomics approach
Q-SAR
e.g. EST
Combinatorial testing pipe line for DART shows high predictivity to mammalian data
Standard operational protocols for exposure
Internal dose assessment after exposure
Omics: transcriptomic, and kinomic analysis and RNAi to identify mode of action
Dose-response curves
Phenotypic data alignment
Validation of the test system
Compound exposure
Shell International B.V.
APPLICATION OF A NOVEL, PATHWAY-BASED SCREEN FOR DEVELOPMENTAL AND REPRODUCTIVE TOXICITY
Chantal Smulders ,
Shell
April 2016
Shell International B.V.
INDUSTRY VALUE OF DART SCREEN & 3R BENEFITS
April 2016
Product development pipeline
early selection of promising substance
avoidance of animal testing multiple new substances
Support read-across strategies and categorization
maximize use of existing animal data
avoidance of additional animal testing
Part of a weight-of-evidence approach
AOP approach allows better prediction of potential human
outcome
Shell International B.V.
APPLICATION OF NOVEL DART SCREEN
April 2016
New process chemicals - 1 existing (Reprox Cat 2)
- 3 structural analogues (A,B,C)
Company name appears here Month 2010Footer
To demonstrate chemical safety, alternative testing and translational toxicology can provide insight in human safety
Alternative testing using high content assays (e.g. genomics) can provide insight in (absence) of potential effects and help elucidate new AOPS
Basis: key pathways for reproduction and development are preserved across species in evolution
Testing in more primitive, less sentient species can provide insight in potential perturbance of human pathways
Major reproductive and developmental toxicity phenotypes in mammals (humans) can be predicted
Monday, July 27, 2015
DEVELOPMENT OF A NEW SCREENING ASSAY
Shell International B.V. April 2016
ASSAYS DEVELOPED IN NC3RS PREDART CRACK-IT PROJECT
Collaborative project between Shell and Syngenta
Aim to assess biomarker development in a panel of in vitro and in vivo alternative test species:-
amoeba (Dictyostelium discoideum),
nematode (Caenorhabditis elegans)
zebrafish (Danio rerio)
and embryonic stem cells)
Assess if these can enable reliable prediction of potentially hazardous compounds taking advantage of evolutionary conserved molecular pathways across the test species
15
Shell International B.V.
INDUSTRIAL APPLICATION OF THE TEST SPECIES: EXPOSURE TO A DART CHEMICAL THAT SHOULD INDUCE
DART ONLY AT A HIGH DOSE: DEVELOPMENTAL DELAY
April 2016
Compound B
A.
B.
C. D.
4 dpf control
4 dpf 10mM PIP
Adult control10mM PIP
Existing
Existing
Shell International B.V.
ANALOGOUS COMPOUND ASSESSMENT INDICATESDEVELOPMENTAL DELAY MOST STRONGLY AT HIGHER
DOSE
April 2016
0102030405060708090
100
zebrafish
nematode
Trip-Hep (AVG)
A. B.
C. D.
Piperazine 1‐(2‐Hydroxyethyl Piperazine)
1,4‐Bis(2‐Hydroxyethyl)piperazine
concentration
concentration concentration
concentration
% Develop
men
tal delay
% Develop
men
tal delay
% Develop
men
tal delay
% Develop
men
tal delay
0102030405060708090100
zebrafish
nematode0
102030405060708090100
Control (H2
O)
1µM
10µM
100µ
M
1mM
10mM
zebrafish
nematode
0102030405060708090100
zebrafish
nematode
Existing A
B C
Shell International B.V.
PERCENTAGE OF AFFECTED ZEBRAFISH LARVAE -TERATOGENIC EFFECTS
April 2016
ExistingABC
Number of fish
Shell International B.V.
C. ELEGANS: EFFECTS ON BROOD SIZE ONLY AT HIGH CONCENTRATIONS
Control 0.1µM 1µM 10µM 100µM 1mM 10mM
Existing - - - - *x *x *xA - - - - * * *xB - - - * * - *C - - - - - - -
April 2016
* statistically significant effects on brood size compared to controlx statistically significant effects on development compared to control
Shell International B.V.
SUMMARY RESULTS DART SCREEN
April 2016
Reproductive toxicity effects in nematodes and zebrafish only observed at high concentrations (100 µM and above)
These concentrations are unlikely to be reached in vivo in humans under conditions of use.
The existing substance - showing reproductive effects at high concentration only - was the most potent reproductive toxicant to nematodes and zebrafish.
These results are in alignment with the reported test data for rats and rabbits on this substance, where indications of reproductive effects were only observed at high test concentrations and are considered to be a consequence of maternal toxicity rather than a direct reproductive effect
The structural analogs tested are unlikely to be developmental toxicants in humans.
Shell International B.V.
CONCLUSIONS USABILITY DART SCREEN
April 2016
The results of the zebrafish and nematodes are in alignment with data obtained from mammalian toxicity studies, indicating that these have potential as developmental toxicity screens
Despite not replacing a reprotoxicity study in animals one-on-one, significant reduction of animal testing can be achieved
Duration and cost of the DART screen allows application early in the product development pipeline
Further mapping of developmental AOP across species is needed
In vitro – in vivo dose extrapolation is key for successful use of the screen in risk assessment
If care is taken regarding relevance of exposure pathways useful environmentally relevant (ecotoxicological) data can also be obtained
Acknowledgements
At what stage would you be willing to invest money when using the alternative test models for DART testing?
1. Now. They can already be used for pre-screening of large compound sets.
2. Not yet. More data/information is required before I trust these systems.
3. Only when they are officially accepted by the regulators.
4. Never
Now.
They
can a
lread
y b...
Not y
et. M
ore da
ta/inf
...
Only
when
they
are o
ffici...
Neve
r
31%
4%
19%
46%
Interactive question 1
Please contact wildwaterm@gmail.com for more information
In principle, would your company/institution be willing to join the effort to help us to further validate these organismal 3Rs models?
1. Yes, we could provide compounds
2. Yes, compounds with mammalian data
3. Yes, investment (time, money and/or resource) to enable testing in parallel to mammalian studies
4. Yes, investment (time, money and/or resource) to pre-screen chemicals to inform choices for further product development (e.g. prioritisation)
5. No
6. Unable to comment
Yes,
we co
uld pr
ovide
c...
Yes,
comp
ound
s with
...
Yes,
invest
ment
(time
, m...
Yes,
invest
ment
(time
, m... No
Unab
le to
comm
ent
12% 12%
54%
7%9%6%
Interactive question 2
top related