Report date: January 28, 2015
Hamner TT21C: Toxicity Pathway and Network Biology Program Review
Dec 8 & 9, 2014
The Hamner Institutes for Health Sciences held their annual TT21C research program review on Dec
8 & 9, 2014. The format, similar to last year, had posters, a reception and dinner the first day with
presentations and discussions of our three case studies – PPARα signaling, estrogen-mediated cell
proliferation and p53-DNA damage pathways – the second day. The formal presentation period was
followed by a session with the Hamner TT21C leadership team and attendees to discuss specifics of the
2015 research planning and overall strategic vision for the program.
Our TT21C program develops fit-for-purpose toxicity pathway assays to accelerate implementation of
key recommendations from the 2007 National Research Council (NRC) report, “Toxicity Testing in the
21st Century: A Vision and a Strategy”. The three case studies serve several purposes. They examine
the role of mechanistic dose-response modeling of cellular responses in contemporary safety
assessments; develop improved understanding of modes of action at the cellular level; and improve the
suite of assays to link molecular initiating events on through to adverse outcome pathways. In our
usage, “fit-for-purpose” indicates a human cell-based assay that provides measures of response and
tools for interpretation such that the results from the assay will be sufficient for chemical safety
assessment without resorting to in-life animal studies. We have discussed the rationale for our case
studies early in the development of our research planning (Andersen et al., 2011).
The Hamner invited sponsors and other interested parties to the review to discuss the status of the
three primary case studies. Current sponsors are Dow Chemical, Dow Corning Corporation, ExxonMobil
Foundation, Unilever, CropLife America, DuPont, 3M and Agilent Technologies. The twelve posters for
discussion were on display throughout the meeting and showed progress with data collection for
moving the case studies toward risk assessment applications and also highlighted the more generic tools
developed at Hamner to support dose response assessments from the in vitro studies. At this year’s
Society of Toxicology meeting in San Diego, Hamner PIs and staff will present eight
presentation/posters. Titles and abstracts for these posters are in Appendix A. In addition, the material
provided to participants at the review – including the agenda, poster titles with abstracts, and the list of
attendees – are in Appendix B. Interested parties can download the talks, posters and key 2014 TT21C
papers/chapters from the Hamner website (http://www.thehamner.org/tt21c-meeting). The files are
protected: please contact Geoff Bock at The Hamner (gbock@thehamner.org) for the password.
In addition to the talks by Hamner staff, Dr. Mary McBride, Director, Governmental Relations, Life
Sciences & Chemical Analysis, Agilent Technologies, Inc. provided a summary of a workshop developed
jointly by Agilent and Hamner and presented in China last year. The workshop course, “Systems
Toxicology: Developing Mechanistic Understanding of Toxicity Pathways and Establishing Predictive
Toxicology for Use in Risk Assessments”, was held in Beijing, China Oct 14-16, 2014. The first day
consisted of a series of plenary presentations to a larger audience of participants across multiple sectors
– academics, businesses, and government agencies. On Days 2 and 3, four courses were run
concurrently with 15 to 30 participants per course. The courses were (i) in vitro methods, (ii)
genomics/transcriptomics, (iii) metabolomics, and (iv) computational systems biology. Hamner staff –
Drs. Andersen and Clewell - provided two of the talks in the plenary session. Dr. Clewell led the in vitro
methods course and Drs. Qiang Zhang, Patrick McMullen and Mel Andersen, all from The Hamner, were
leads in the computational systems biology course. Overall the program was well received and there is
keen interest in China to learn more about systems toxicology and its possible applications to support
predictive safety assessment methods for commercial products.
Our efforts to develop fit-for-purpose assays have also received a high degree of interest from Europe.
Dr. Maurice Whelan, Director, Systems Toxicology, the Joint Research Centre, Ispra, Italy and also the
Director, European Center for Validation of Alternative Methods, is a co-editor for a forthcoming book,
“Validating Alternative Methods for Toxicity Testing”. A team of Hamner and Unilever authors
contributed an invited paper to this volume (Clewell et al., 2015). On reading our contribution, Dr.
Whelan responded in an e-mail:
“Many, many thanks for delivering this chapter. It was no easy task to write I'm sure but what a
result! I have only read through it once but that was enough to appreciate what a milestone this
chapter will prove to be, and how it redefines validation in the context of new pathway-based
approaches to toxicology. Others have hinted at this, including myself, but you guys have shown it in
action through utterly convincing case studies. Hats off to you.”
We are pleased that our TT21C case studies are gaining visibility and showing a path forward for the use
of new methods for safety assessment. In addition, the emphasis on a systems approach to
understanding pathway function has provided improved linkage of our cell-based assays to modes-ofaction and are contributing tools to deal with several key questions in current risk assessment, including
the relevance of rat/mouse liver responses for human risk assessment, whether DNA-damaging
compounds have threshold responses at low doses, and the adequacy of simple estrogen receptor
binding assays for assessing endocrine disruption. The following text briefly summarizes project status
as reported at the meeting and mentions the contributions of the work for more immediate
applications.
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Estrogen Signaling in the Uterus: The overall flow chart for our
case studies (Figure 1) shows the steps from problem
formulation to development of the fit-for-purpose assay and
then moving on down to pathway based safety assessments. In
addition to efforts on specific pathways, programmatic research
on genomics, high-content imaging, computational pathway
modeling and in vitro to in vivo extrapolation are integral parts
of bringing individual case studies through to safety
assessments. These programmatic capabilities are not directly
noted in this report, but are described in several of the papers in
the reference list (Trask et al., 2014; Yoon et al., 2012; Zhang et
al., 2013, 2014, 2015).
With our estrogen case study we have continued developing the
proliferation assay using the Ishikawa cells – a line derived from
Figure 1. Hamner pipeline for developing pathway-based
a human uterine adenocarcinoma. With these cells, we have safety assessments.
conducted a range of genomic and functional assays to look at
the role of various estrogen-responsive receptors ERα66; ERα46; ERα36 and GPER (G-Coupled Protein
Estrogen Receptor) in controlling proliferation, the phenotypic response used as our measure of
adversity. All these studies are conducted in a dose response manner to evaluate responses across a
large dose range. The work in 2014 focused on evaluating literature studies on these receptors,
research unraveling the role of these receptors in Ishikawa cell proliferation and initiating work to
develop a computational network model for proliferation.
The body of work on these receptors produced a
working schematic for the role of each receptor in
proliferation (Figure 2).
Here we show the
interconnections of the network and the positive
interactions (arrows) and the negative interactions
(connections ending with a perpendicular line). With
GPER, we saw that its concentration is increased
following EE treatment (as noted in the heat map).
Increasing doses of a GPER selective ligand (G1) were
also found to increase proliferation (lower right). The
overall pathway structure has negative regulators
Figure 2. Presumed role of GPER in proliferative responses to (ERα46 and ERβ), positive regulators (ERα66 and
Ethinyl Estradiol and model schematic for its actions.
ERα36) and a non-linear EGF kinase cascade that
activates proliferation. Our results currently indicate
that the treatment of cells with EE renders the cells more responsive to proliferation. An electrical
circuit analogy is that the EE treatment lowers the resistance in the uterine receptor-tyrosine kinase
pathway activated by epidermal growth factor and similar ligands.
At present the high throughput assays used by the US EPA ToxCast program focus almost exclusively on
assays for ERα66 and on early pathway responses, such as ligand binding and transactivation of reporter
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systems. They do have one cell-based proliferation assay with a breast cancer cell line. Our estrogen
pathway case study emphasizes the need to develop more comprehensive assessment of the various
receptors and a focus on the ‘adverse’ responses rather than relying on just more simple pathway
perturbations – binding, transactivation, etc. We are completing one paper on the development of the
fit-for-purpose proliferation assay in uterine cells and another that reviews current knowledge on the
network that controls estrogen mediated proliferation.
PPARα Pathways in Primary Hepatocytes and Rat Liver: Our paper mapping the transcriptional
regulatory network in primary human hepatocytes appeared in Chemical and Biological Interactions
(McMullen et al., 2014). Over the course of 2014, we completed work evaluating differences in the
PPARα network between humans and rats, established the likely species-specific biology leading to the
difference, compared responses of rat hepatocytes in vitro and in vivo in the intact liver, and completed
studies tentatively identifying metabolites secreted from hepatocytes following treatment with the
selective PPARα agonist GW7647.
The number of genes altered by GW7647 in rat
hepatocytes was much greater than in humans
and a larger number of these were
downregulated (Figure 3A). As shown in the
bubble map of functional categories (Figure 3B),
the downregulated genes in yellow were
involved in processes such as apoptosis, wound
healing and cytoskeletal reorganization. There
was good correspondence in the core metabolic
pathway for fatty acid, lipid and amino acid
metabolism for the two species (shown in blue).
Because there is less information on DNA-binding
sites in rats compared to human or mouse, we
could not create the transcriptional network as
we had done in humans (Figure 3C). However,
the time course of gene expression showed that
several key liver transcription factors were
decreased after PPARα activation in rats –
notably Ets1 and Hnf-6. Finally, we examined the
binding regions for PPARα in differentially
regulated genes that contained a PPARαresponse element. For the down regulated
genes, we noted that the flanking regions Figure 3. Toxicity pathway studies in action. We have used gene
required for activation and interaction lacked expression microarray experiments (A) and other data streams to map
PPARα regulation in humans and rodents (B-D).
some of the consensus nucleotides involved in
RXR binding (Figure 3D). This downregulated
network, present in rat and not human, appears to prime the hepatocytes for proliferation. It is likely
that the response to nuclear receptor activation in rodents arises from this secondary pathway that is
absent in humans. It will be important to see if this response patterns holds for other receptors forming
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heterodimers with RXR, e.g., FXR, PXR, CAR, and LXR. Our work indicates that the human-rat differences
are qualitative based on differential receptor networks in the two species.
Our metabolomic analysis of the media from GW7647-treated hepatocytes showed similarities in classes
of molecules produced in rats and humans. These studies need to be improved to allow unambiguous
identification of the metabolites. Tentative analysis indicates the presence of several important
signaling molecules – fatty acid glycine conjugates and both lyso- and cyclic phosphatidic acids (LPA and
CPA). The conjugates or their precursor ethanolamides signal through PPARα and cannabinoid
receptors. LPA and CPA activate specific GPCR pathways. These results indicate that release of GPCRactive small molecules may take part in paracrine signaling in liver and in extrahepatic tissues. Based on
discussions at the meeting, we are making some changes to the human-rat comparison paper and
preparing its submission later this month. The metabolomics results will be published in a later paper
focusing on improved identification of the features by HPLC-MS-MS. We are contracting these studies
with Stemina Biomarker Discovery, Inc. in Madison, WI.
Figure 4. Benchmark dose values for
various DNA damage endpoints in
response to three genotoxins. Micronuclei
formation occurs at a lower concentration
than transcriptional activation (purple and
orange markers, respectively).
p53-DNA Damage Pathway Studies: Unilever has supported our
project on the p53-DNA damage pathway. Our first papers have
outlined the development of fit-for-purpose assays to look at multiple
biomarkers of response across a range of doses (Sun et al., 2013;
Adeleye et al., 2014; Clewell et al., 2014). The initial working hypothesis
regarding low dose adaptation through upregulation of repair genes
was not consistent with the early results showing that changes in gene
expression occurred at doses higher than concentrations causing
increases in micronuclei formation – our marker for adversity in these
assays. The dose response for various endpoints with three of our
compounds - etoposide (ETP), quercetin (QUE) and methylmethanesulfonate (MMS) - showed that transcription (Figure 4, orange marker)
was less sensitive than micronuclei formation (purple marker). Despite
the large numbers of markers we evaluated, none had benchmark dose
(BMD) values less than the BMD for micronuclei. These observations
have led to a shift in focus of our experiments to look at formation of
DNA-repair centers (DRCs).
DRCs are visualized in the nucleus after DNA-damage as multiple bright spots that can be resolved into
individual foci and counted. The image in Figure 5 shows the response after a very high dose (400
µg/ml) of neocarzinostatin (NCS). DRCs contain several phosphorylated proteins – p53 binding protein,
γ-H2AX - recruited to sites of damage in order to facilitate repair. Using high content imaging, the
formation and resolution of these DRCs can be measured and quantified. At low doses, the time course
of DRC formation (Figure 5, bottom left) shows that DRCs form and resolve quickly. At the higher dose
(bottom right), more DRCs form and they are more persistent, not resolving over the 24-hour
observation period. Our results show that at doses below those forming micronuclei, there are active
processes working to control low to moderate levels of DNA-stress. When the level of damage
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Figure 5. Schematic of our mathematical model for DNA repair center
formation and resolution.
continues to increase, DRC formation and DNArepair processes cannot keep up with damage
and cell division leads to permanent DNA-damage
in the form of micronuclei. Several Hamner staff
members are working on a computational
pathway model for DNA-damage repair based on
our kinetic studies of DRC time course with both
NCS and ETP (Figure 5). ETP inhibits repair of
double strand breaks and has different kinetics of
DRC formation/resolution. Our work strongly
indicates that even DNA-reactive compounds
may have threshold responses at low doses.
These studies and the resulting computational
model will be described in manuscripts to be
completed in 2015.
Summary/Plans for 2015: The technical portion of the meeting emphasized the progress in completing
the key components needed to move the three projects to a proof of concept stage for safety
assessments. We also noted the value for contemporary issues in risk assessment already derived from
the refinement of mode-of-action studies at the cellular level for each of these pathways. An important
activity this year will be to write papers and present material at meetings to show the progress and the
promise of these fit-for-purpose assays for safety assessments. On the other hand, the early success
helps in selecting other pathways that may be ripe for evaluation, e.g., other liver nuclear receptor
pathways or stress pathways. With limited resources, we have to carefully balance the workload of new
studies against taking the time/effort to communicate the value of the work we have completed.
The question of the directions of the projects/program for 2015 was also discussed in a small roundtable
with sponsors/attendees at the end of the presentations. Hamner participants were Drs. Mel Andersen,
Rebecca Clewell, and Patrick McMullen. There were good discussions about the next steps and the
overall commentary deserves more space than a short paragraph here. However, the key message was
on the importance of getting the results to press quickly for all the case studies. One suggestion was to
do a publication combining all three case studies into one paper. The consensus seemed to be to get
the individual examples to press quickly and do a semi-review of the three together as a second step.
We also heard a note of caution in trying to move to increase the portfolio of pathways too quickly.
Communication of results and education of stakeholders on the value of the investment in the program
are critical for 2015. We also heard that Hamner should begin to consider how this work could lead to
new assays for customers; consider how the work reduces uncertainties in risk assessment; conduct a
gap analysis on safety assessment needs; and, consider how we market the advantages of the mode-ofaction tools for current issues in risk assessment not just for future approaches using cells in culture.
We very much appreciated the thoughtful comments and constructive dialog from the participants.
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References:
Adeleye, Y., Andersen, M., Clewell, R.A., Davies, M. Dent, M., Malcomber, S., Nichol, B., Westmoreland,
C., White, A. and Carmichael, P. (2015). Implementing toxicity testing in the 21st century (TT21C):
making safety decisions using toxicity pathways, and progress in a prototype risk assessment.
Toxicology. Feb 25. pii: S0300-483X(14)00032-8. doi: 10.1016/j.tox.2014.02.007.
Andersen, M.E., Clewell, H.J., III, Carmichael, P.L. and Boekelheide, K. (2011). Food for Thought……Can a
Case Study Approach Speed Implementation of the NRC Report: “Toxicity Testing in the 21st
Century: A Vision and A Strategy”. ALTEX, 28, 175-182.
Clewell, R.A., Sun, B., Adeleye, Y., Carmichael, P., Efremenko, A., McMullen, P.D., Pendse, S., O. J. Trask,
O.J., White, A., and Andersen, M.E. (2014). Profiling DNA damage pathways activated by chemicals
with distinct mechanisms of action. Toxicol. Sci., 142, 56-73.
Clewell, R.A., McMullen, P.D., Adeleye, Y. and Andersen, M.E. (2015). Pathway Based Toxicology and Fitfor-Purpose Assays. In “Validating Alternative Methods for Toxicity Testing. Chandrta Eskes and
Maurice Whelan, Eds. Springer International Publishing AG, Cha (volume in preparation).
McMullen, P.D., Bhattacharya, S., Woods, C.G., Sun, B., Ross, S.M., Miller, M.E., McBride, M.T., LeCluyse,
E.L., Clewell, R.A. and Andersen, M.E. (2014). A map of the PPARα transcription regulatory network
for primary human hepatocytes, Chem. Biol.-Interactions, 209, 14-24.
Sun, B., Ross, S.M., Trask, O.J., Carmichael, P., White, A., Andersen, M.E. and Clewell, R.A. (2013).
Assessing differences in DNA-damage and Genotoxicity for two polyphenol, flavonoids - Quercetin
and Curcumin. Toxicology in vitro, 27, 1877-1887.
Trask, O.J., Jr., Moore, A. and LeCluyse, E.L. (2014). A micropatterned hepatocyte coculture model for
assessment of liver toxicity using high-content imaging analysis. Assay and Drug Dev. Technol., 12.
16 -27. doi: 10.1089/adt.2013.525. Epub 2014 Jan 20.
Yoon, M., Campbell, J., Andersen, M.E. and Clewell, H. J. (2012). In vitro to in vivo extrapolation of cellbased toxicity assay results. Crit Rev Toxicol., 42, 633-652.
Zhang, Q., Bhattacharya, S. and Andersen, M.E. (2013). Ultrasensitive response motifs: basic amplifiers
in biochemical network. Open Biology, 2013 Apr24:3(4):130031. Doi: 10.1098/rsob.130031.
Zhang, Q., Bhattacharya, S., Conolly, R.B., Kaminski, N. and Andersen, M.E. (2014). Molecular signaling
network motifs provide a mechanistic basis for cellular threshold responses. Environ Health
Perspect, 122, 1261-1270.
Zhang, Q., Bhattacharya, S., Pi, J., Clewell, R.A., Carmichael, P.L. and Andersen, M.E. (2015). Adaptive
Posttranslational Control of Cellular Stress Pathways in relation to Toxicity Testing and Safety
Assessment. Toxicol. Sci., in review.
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Appendix A
Abstracts for Presentations of Sponsored Projects
Society of Toxicolgy 2015 Annual Meeting
The in vitro and in vivo response to PPARα activation in rats
Patrick D. McMullen, Salil Pendse, Sudin Bhattacharya, Edward L. LeCluyse, Rebecca A. Clewell, and
Melvin E. Andersen, The Hamner Institutes for Health Sciences, Research Triangle Park, NC
In vivo, sustained PPARα activation leads to tumors in rats, but not humans. To better understand this
mechanism, we cultured primary rat hepatocytes and exposed them to the PPARα-selective agonist
GW7647 over a range of 5 concentrations (0.001–10μM) and assayed gene expression via microarray
after 5 intervals of exposure (2–72h). Additionally, we performed an in vivo study in which male rats
were exposed to GW7647 via oral gavage for 4 days across three doses. Analytical chemistry confirmed
that GW7647 serum concentrations of treated animals are dose dependent and complementary to the
concentrations used for our in vitro experiments. We observed a dose-dependent increase in liver
weight in treated animals, but serum ALT levels were only slightly increased, indicating the absence of
overt hepatotoxicity. Cell division was increased in the livers of treated animals.
Lower doses of GW7647 that did not cause significant increases in proliferation in vivo produced gene
expression profiles that were similar to those observed at in vitro concentrations of 1μM, showing
upregulation of β-oxidation enzymes and other lipid metabolism machinery. High in vivo exposures,
however, led to activation of additional biological functions, including genes associated with mitosis and
cell-cycle checkpoint control. Immunohistochemical staining suggests that induction of peroxisomespecific markers in response to PPARα activation is primarily a centrilobular phenomenon. We used laser
capture microdissection to separate periportal, midzonal, and centrilobular tissues from GW7647treated rats and are using microarrays to determine regional differences in the response to PPARα
activation. Purified rat hepatocytes do not proliferate in response to PPARα activation, supporting the
model that hepatic proliferation requires mitogenic signaling from non-parenchymal cells. We are using
this information to design better in vitro testing strategies for nuclear-receptor-mediated proliferation.
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Estrogen Receptor isoforms ERα66, ERα46, and ERα36 exhibit distinct signaling
interactions during Estrogen-mediated proliferative events
Michelle M Miller, Rebecca Alyea, Jian Dong, Melvin E. Andersen, and Rebecca A. ClewellThe Hamner
Institutes for Health Sciences, Research Triangle Park, NC
Traditional testing paradigms for endocrine disruptor screening are being challenged as the future of risk
assessment moves towards fit-for-purpose in vitro assays to predict adverse outcomes. To replace
current in vivo assays, an understanding of the key events in estrogenic signaling must be achieved. To
this end, human endometrial Ishikawa (IK) cells, previously shown to demonstrate phenotypic responses
to estrogens, were used to examine the role of estrogen receptor (ER) isoforms in estrogen-mediated
proliferation. IK cells constitutively express all five of the known ERs: ERα66, ERα46, ERα36, ERβ, and
GPER, (measured by PCR and western). We used selective ER agonists to evaluate the role of ER
isoforms in proliferation. Agonists for ERβ (DPN) and GPER (G1) did not induce proliferation. However,
ERα-specific agonist (PPT) induced robust proliferation, indicating ERα is the main receptor mediating
these events. Since PPT may bind all three ERα isoforms, we next asked if the isoforms had unique
contributions to uterine cell proliferation. We designed overexpression systems by inserting lentiviral
constructs encoding ERα-GFP fusion proteins specific for each isoform in IK cells. High-content imaging
confirmed expression of the proteins. Preliminary analyses indicate overexpression of ERα66 or ERα36
increases baseline proliferation in IK cells and ERα66 further increased both sensitivity and proliferative
response to ethinyl estradiol (EE). ERα46 overexpression, however, correlated with a significant
reduction in EE-induced proliferation. These data suggest that while ERα66 and ERα36 initiate
proliferative signaling, ERα46 may antagonize EE-induced proliferative pathways. Furthermore these
studies demonstrate the importance of non-canonical ER signaling in proliferation and highlight the
need for screening assays that incorporate key biological processes driving chemical induced estrogenic
responses rather than simply focusing on ER66 binding or E2-mediated transactivation.
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Computational Systems Biology Modeling of DNA-damage Stress Pathways for
Assessing Mutation Rates at Low Doses
Rebecca A. Clewell1, Salil Pendse1, Patrick McMullen1, Paul Carmichael2, Yeyejide Adeleye2, Melvin E.
Andersen1. 1The Hamner Institutes for Health Sciences, Research Triangle Park, NC and 2Unilever,
Bedfordshire, UK
Homeostasis by cellular stress response pathways involves negative feedback acting through a series of
steps. Many stress response pathways have rapid response, post-translational signaling and slower
signaling through transcriptional upregulation. We examined multiple biological read-outs in a human
cell line (HT1080) treated with several DNA-damaging compounds to support mechanistic computational
modeling for micronuclei (MN) formation across wide dose ranges. The readouts included dose and
time-dependent whole genome gene expression, DNA repair center (DRC) formation through high
content imaging of pH2AX and p5BP1, as well as measures of key proteins in the p53 pathway and MN.
Transcriptional upregulation only occurred at concentrations with clear increases in MN formation.
Post-translational activation of DNA-repair processes acting through specific kinases appears to be the
main contributor to regulation of DNA-damage at lower doses. We have developed computational
pathway models to describe the relationship between DRCs and MN formation for two potent double
strand break inducers with very different MN dose-response curves: etoposide (linear) and the gamma
irradiation mimic neocarzinostatin (threshold-like). These models, ranging from simple empirical
descriptions of the data to more biologically oriented descriptions of the homeostatic feedback loops,
provide a quantitative framework for assessing the key processes governing MN prevention at low doses
of different types of DNA damaging chemicals. Ultimately, these models will support decisions for in
vitro only risk assessments, by providing a quantitative description of how low dose threshold behavior
may be achieved in mutation response, and helping define concentrations leading to cellular adaption
and potential adversity.
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Development of an In vitro High-Content Imaging Assay for Quantitative
Assessment of Mouse Hepatocyte Proliferation
Valerie Soldatow1, Richard Peffer2, David Cowie2, Rebecca Clewell1, Melvin Andersen1, Edward LeCluyse1,
and Chad Deisenroth1, 1The Hamner Institutes for Health Sciences, Research Triangle Park, NC and
2
Syngenta Crop Protection, Greensboro, NC
The two-year rodent bioassay is the standard metric for determination of carcinogenicity from exposure
to synthetic or natural chemicals. This approach is labor- and cost-intensive with limited throughput for
assessing hepatocarcinogenesis of novel chemicals with different modes of action. Transition to an in
vitro model has been hampered by challenges with reproducing proliferative responses in cultured
primary hepatocytes. Using an array of recombinant growth factors, cytokines, and model CAR
activators, an effort was made to develop an in vitro high-content imaging based assay for quantitative
assessment of nascent DNA synthesis in primary cultures of CD-1 mouse hepatocytes. Detection of DNA
synthesis was performed using click chemistry labeling of the nucleoside analog 5-ethynyl-2′deoxyuridine (EdU). Optimization of the DNA labeling index revealed time- and seeding densitydependent effects to growth factor induced DNA synthesis. Hepatocytes were responsive to the CYP
induction properties of known CAR activators such as TCPOBOP, but were unresponsive for DNA
synthesis. Subsequently, the proliferative responses to growth factors EGF, HGF, and TGF-α were
evaluated and optimized. Additional supplementation with the cytokine IL-6 significantly increased DNA
synthesis by each of these growth factors. The assay was multiplexed to enable direct quantitation of
DNA synthesis, cytotoxicity, and cell count endpoints. Using the optimized defined media cocktail, the
EdU labeling response was enhanced in TCPOBOP treated hepatocytes in a concentration-dependent
manner. The results demonstrate that cofactors normally available to hepatocytes via non-parenchymal
cells enhance the induced DNA synthesis response to a CAR activator.
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Characterization of Estrogenic Responses in Human Endometrial Primary
Epithelial and Carcinoma Cell Lines
Pergentino Balbuena, Rebecca Alyea, Michelle Miller, Susan M. Ross, Sean Rowley, Melvin E. Andersen,
Rebecca A. ClewellThe Hamner Institutes for Health Sciences, Research Triangle Park, NC.
Development of fit-for-purpose in vitro assays to study cellular signaling and functional response is
essential for characterizing toxicity pathway structure and measuring adverse perturbations by
exogenous chemicals. To identify the most appropriate model for evaluating effects of estrogenic
compounds on uterine cells, we compared the ability of several in vitro models to recapitulate the
proliferative response to ethinyl estradiol (EE) observed in vivo. Human endometrial epithelial primary
cells (HEEC), human uterine epithelial adenocarcinoma cells (Ishikawa, IK), human breast epithelial
carcinoma cells (MCF7), and a co-culture of IK and endometrium stromal cells were examined.
Proliferation in response to EE was observed in all the in vitro models with the MTS assay. The primary
uterine cells (HEECs) demonstrated the greatest induction, with a maximum of 1.8-fold change and a
lowest effective concentration of 10 pM. The three cancer cell lines showed similar levels of induction
(~1.2-fold). MCF7s were most sensitive to EE, however, inducing proliferation at lower doses than HEEC
or IKs. Expressions of five estrogen receptors (ERs) that modulate proliferative signaling were evaluated
to help identify the basis of the differential response. Both MCF7 and IKs express full length estrogen
receptor ERα (ERα66), its isoforms ERα36 and ERα46, the estrogen binding G-coupled protein receptor
(GPER) and ERβ proteins. MCF7s, however, showed a much higher expression of ERα66. Surprisingly,
while ERα36, ERβ and GPER mRNA and proteins were observed in HEECs, only very low levels of ERα66
and ERα46 mRNA were found. Lack of ERα66 protein in HEECs shows that estrogen mediated
proliferation can be induced independently of full-length ERα66 and that non-canonical pathways may
be important in the uterus. These results could have wide-ranging implications for better assay design,
as current high throughput screening efforts focus solely on ERα66 activation in breast cancer cell lines
to predict estrogenic responses.
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Oxidative Stressors Induce Differential Cellular Responses between Human
Primary and HaCaT Keratinocyte Cells
Bowen Huang1, Peng Xue1, Bin Sun1, Jingbo Pi1, Andy White2, Melvin E. Andersen1, and Rebecca A.
Clewell1. 1The Hamner Institutes for Health Sciences, Research Triangle Park, NC and 2Unilever,
Bedfordshire, UK
Oxidative stress reflects the status of imbalance between production of reactive oxygen species (ROS)
and the ability of the system to cope with ROS. Nuclear factor E2-related factor 2 (Nrf2) plays a critical
role in ROS defense through regulation of a battery of genes coding antioxidant proteins and
detoxification enzymes by binding the antioxidant response element (ARE) and initiating gene
transcription. We studied the Nrf2-response pathway and determined doses of prototype chemicals
(curcumin, hydrogen peroxide; H2O2) associated with sub-threshold, adaptive and adverse oxidative
cellular responses in two cell types: immortalized keratinocytes (HaCaT) and primary neonatal
keratinocytes (HEKn). Our goal is to determine the key biomarkers in the pathway and identify regions
of safety for oxidative stressors. In HaCaT cells, markers of ROS and adaptive antioxidant responses —
Nrf2 protein accumulation, ARE transactivation, and expression of antioxidant genes — were induced by
H2O2 and curcumin, but no significant oxidative damage was observed in assays for protein oxidation,
protein nitration, or DNA oxidation (8-hydroxy-2’-deoxyguanine;). However, both compounds induced
strong increases in protein nitration (5-fold over control) and carbonylation at non-cytotoxic
concentrations in the HEKn cells, indicating that HEKn cells are more sensitive to oxidative damage than
HaCaT cells, and may therefore be the more conservative in vitro model for oxidative stress response. In
an effort to advance the transition to in vitro toxicity testing described in the National Academies of
Sciences report, “Toxicity Testing in the 21st Century (TT21C): A Vision and A Strategy”, these time- and
dose-response data are being used to support development of a computational model for oxidative
stress response and, ultimately, provide proof of concept in vitro based safety assessments.
13
Evaluation of Dose-Dependent DNA Repair Center Kinetics and Micronucleus
Induction in Chemicals Causing Different Types of DNA Damage
Bin Sun1, Joe Trask1, Sean Rowley1, Paul L. Carmichael2, Yeyejide Adeleye2, Melvin E. Andersen1, and
Rebecca A. Clewell1 1The Hamner Institutes for Health Sciences, Research Triangle Park, NC and
2
Unilever, Bedfordshire, UK
DNA repair centers (DRCs) are aggregates of repair proteins that bind sites of double strand breaks
(DSBs). Left unrepaired, these temporary DSBs may be converted to micronuclei (MN) – small pieces of
DNA lost during replication. We studied dose-dependent DRC kinetics in an effort to better understand
how these repair processes affect the shape of the response curves for MN induction. Studies were
performed in human fibrosarcoma cells with native p53 (HT1080). Previously, we found that both
etoposide (ETP, topoII inhibitor) and neocarzinostatin (NCS, IR mimic) caused rapid DRC formation (< 2
h). At low doses, these DRCs were more efficiently resolved (repaired) with NCS compared to ETP. This
difference in DSB repair may explain why the dose-response curve for MN induction is threshold-like for
NCS, but linear for ETP (i.e., efficient vs. poor repair of DSBs). The current study examined DRC kinetics
in three genotoxic chemicals with distinct mechanisms: methylmethane sulfonate (MMS, alkylation),
mitomycin C (MMC, crosslinking) and H2O2, (oxidation). Similar to NCS, H2O2 induced a rapid DRC
response followed by rapid resolution at low doses (≤ 100 µM), consistent with the threshold-like MN
curve. In contrast to the other chemicals, MMS and MMC did not induce DRCs until 24 h, which is
consistent with the fact that they only cause DSBs indirectly (misrepaired lesions). Induction of DRCs was
effectively prevented at low concentrations (≤ 60 µM) of MMS, indicating that a distinct DNA repair
process prevents DSBs at low doses leading to observed threshold behavior for MN. MMC, however,
showed a linear increase in DRCs and a failure of these DRCs to resolve, consistent with poor repair of
both initial lesions and subsequent DRCs, as well as the linear MN curve. Taken together, our results
indicate that DRC kinetics can help to interpret the shape of dose-response curves for genotoxicity.
14
A multi-scale mechanistic model of TCDD-induced toxicity for assessing
expected dose-responses for adverse outcome pathways in liver
Sudin Bhattacharya, Patrick McMullen, Salil Pendse and Melvin E. Andersen, The Hamner Institutes for
Health Sciences, Research Triangle Park, NC
Ongoing efforts to describe and apply adverse outcome pathways (AOPs) for risk assessment require
linkage of early molecular interactions to phenotypic responses. New advances in multiscale “virtual
tissue” models provide a quantitative framework to link together: (a) tissue disposition of a chemical; (b)
molecular initiating events such as receptor binding; (c) explicit models of intracellular and organism
level AOPs; and, (d) phenotypic endpoints like cell proliferation. Here we describe our development of a
virtual tissue model of the rodent liver lobule for activation of the aryl hydrocarbon receptor (AhR)
toxicity pathway in hepatocytes exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This case study
shows the process of data integration and multiscale model development for mechanistic dose-response
prediction and application of the AOP framework to risk assessment. First, a modified cluster
aggregation algorithm provided a two-dimensional computational representation of the rodent liver
lobule. This multicellular lobular model was linked to the CompuCell3D modeling environment that
incorporated a network representation of intra-hepatocyte activation of the AhR signaling pathway. A
PBPK description of TCDD uptake in the liver through hepatic sinusoids estimated differential cellular
dosimetry as an input to the spatial model. Zonal heterogeneity in TCDD-induced cytochrome induction
arises from for descriptions of spatial gradients in basal AhR expression across the lobule.
Simultaneously, a combination of published gene expression and chromatin immunoprecipitation data
served to refine a detailed transcriptional network of the AhR pathway with differential sensitivity
ascribed for different liver regions. Overall, the multiscale model accurately simulated the observed
dose-responses for various endpoints. Virtual tissue models promise more quantitative linkage to AOPs
than possible through simple narrative descriptions of these processes.
15
Appendix B
2014 TT21C Program Review Materials
Agenda booklet, list of attendees, and titles of talks and posters
16
2014
Annual
Review
TT21C: Toxicity Pathways &
Network Biology Program
December 8&9
Six Davis Drive
Research Triangle Park, NC 27709
+1-919-558-1200
The Hamner Institutes
TT21C: Toxicity Pathways & Network Biology
Table of Contents
Table of Contents
Agenda .................................................................................................................................................................... 2
2014 Program Summary ......................................................................................................................................... 3
Attendees ................................................................................................................................................................. 4
Poster Abstracts....................................................................................................................................................... 5
1.
Safety assessment implications of differential TCDD dose responses across the liver............................... 5
2. Tools to examine differential responses in the liver: a spatial multicellular model of AhR pathway
responses ............................................................................................................................................................. 6
3. Developing an in vitro high-content imaging assay for a measure of adversity - proliferation in mouse
hepatocytes ......................................................................................................................................................... 6
4. Improving the Assay System for Oxidative Stress Responses: Comparison of Human HaCaT and
Primary Epidermal Keratinocyte Cells ............................................................................................................... 7
5. Posttranslational control of adaptive cellular stress responses is an important consideration in toxicity
testing and risk assessment ................................................................................................................................. 7
6. Understanding threshold processes in genotoxicity: Dose response modeling for DNA repair center
formation and resolution ..................................................................................................................................... 8
7. Tools for assaying DNA repair at doses without mutagenic activity: looking at double strand break repair
kinetics in live human HT1080 cells .................................................................................................................. 8
8. Confirming appropriateness of testing in cell lines: Characterization of estrogenic responses in human
endometrial primary epithelial and Ishikawa cells ............................................................................................. 9
9.
Estrogen-mediated proliferation requires contributions from multiple receptors – not simply ERα66 ...... 9
10.
PPARα activation in rats: differential in vivo and in vitro responses influence assay design ................ 10
11.
TT21C at The Hamner: Creating new in vitro tools for quantitative safety assessment ........................ 10
12.
Developing approaches to assess differential regional hepatocyte responses in humans........................ 11
13.
Improving quantitative in vitro to in vivo extrapolation using a liver bioreactor to assess metabolism.. 11
14.
IVIVE modeling and high-throughput tools to advance toxicity testing and risk assessment ................ 12
15. Establishment of sensitive, quantitative and real-time cellular assays for assessment and screening of
modulators of endogenous androgen receptor signaling pathways .................................................................. 12
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The Hamner Institutes
TT21C: Toxicity Pathways & Network Biology
Agenda
Agenda
Monday, December 8, 2014
16:00 – 16:10
Welcome and Introduction
16:10 – 18:30
Poster viewing and reception
18:30 – 19:30
Catered Dinner
Dr. Rebecca Clewell
Tuesday, December 9, 2014
8:00 – 8:30
Breakfast
8:30 – 8:50
Opening remarks and overview of TT21C
program at the Hamner
Dr. Mel Andersen
8:50 – 9:00
Summary of Systems Toxicology
Workshop in Beijing
Dr. Mary McBride
9:00 – 9:30
The Hamner TT21C Safety Assessment
Strategy
Dr. Rebecca Clewell
9:30 – 10:20
Toward a testing strategy for nuclear
receptor-mediated proliferation
Dr. Patrick McMullen
10:20 – 10:35
Break
10:35 – 11:25
Ensuring biological relevance of in vitro
assays for endocrine disruption
Dr. Michelle Miller
11:25 – 12:15
Safety assessment models for genotoxic
chemicals: understanding mechanisms for
threshold dose response
Dr. Rebecca Clewell
12:15 – 13:15
Lunch (Golberg Library)
13:15 – 13:45
Using IVIVE to translate in vitro results to
human exposures
Dr. Harvey Clewell
13:45 – 14:00
Next Steps
Dr. Mel Andersen
14:00 – 15:00
Discussion
15:00
Adjourn
2
The Hamner Institutes
TT21C: Toxicity Pathways & Network Biology
2014 Program Summary
2014 Program Summary
Welcome to The Hamner’s third annual TT21C program
meeting. Here, we provide a short program overview and
attach our 2014 midyear report completed in September. In
close collaboration with our sponsors, we have been
steering our TT21C projects more quickly toward
prototype in vitro assay-based safety assessments
(Figure 1). Our progress in this regard takes a number of
different forms, as our individual projects are at varying
levels of maturity. Collectively, our projects cover a
number of high-level risk assessment issues:
Relevance of animal models for safety assessment. For
about 30% of compounds with reference doses, liver
toxicity serves as the endpoint used for the assessments.
There is great uncertainty about the human relevance of
these liver responses, e.g., increases in liver weight, cell
proliferation, and non-genotoxic carcinogenicity. A major
challenge in chemical risk assessment is the degree to
which liver responses in rodents are relevant for human
safety. These differences become even more problematic
when new in vitro assays in human cells are aligned with Figure 1. Our roadmap for developing prototype risk
legacy studies from rodents. Our work with PPARα assessments. Our TT21C projects span a diverse
signaling focuses on developing in vitro fit-for-purpose complement of stages in this schema.
assays that examine appropriate endpoints in rodent cells
and developing tools needed to use results from similar assays in human cells for safety assessments.
Non-linear responses with genotoxicity. Low-dose linearity—the idea that any exposure to genotoxins
increases risk of mutation—is the curent default used in risk assessment. Our studies on DNA-damage pathways
indicate that thresholds, rather than linearity, are more likely the norm for these stress pathway responses. To
establish stronger experimental and theoretical evidence for the existence of a threshold for genotoxicity, we
have conducted detailed dose-response assessments for several classes of DNA damaging compounds and
developed computational systems biology models to assess regions of safety for exposures to these compounds.
Endocrine disruption. The test battery of the EPA’s Endocrine Disruptor Screening Program focuses on either
binding to full-length ERα66 or subsequent transactivation by the receptor as predictors of endocrine disruption.
However, there are several distinct estrogen receptor proteins. While the manner in which these ER isoforms
contribute to estrogen action is not fully defined, we do know that the full-length ERα66 is not by itself
sufficient to drive cell proliferation. Hamner has developed a variety of in vitro tools to characterize the ERmediated proliferation pathway, to determine the key signaling proteins, including ER isoforms, responsible for
inducing uterine cell proliferation and to create a biological model for proliferation. This body of work provides
fit-for-purpose uterine cell based assays and tools to assess regions of safety for chemicals with estrogenic
activity.
In vitro-to-in vivo extrapolation (IVIVE). An important step in establishing exposure guidelines is a
framework for relating results from in vitro assays to in intact humans. In the coming year, we will leverage
Hamner expertise in pharmacokinetics to develop IVIVE models for commercially-relevant PPARα agonists
(i.e., perfluorinated acids or phthalates) and estrogen receptor agonists (i.e., genistein or alkyl-parabens).
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TT21C: Toxicity Pathways & Network Biology
Attendees
Attendees
Guests
Peter Bent (ACEA)
Mary McBride (Agilent Technologies)
David Fisher (ACC)
Kim Boekelheide (Brown University)
Clare Thorp (CropLife America)
Yvonne Dragan (DuPont)
Katy Goyak (ExxonMobil)
Gary Minsavage (ExxonMobil)
Jim Gigrich (Keysight Technologies)
Tim Pastoor (Syngenta)
Carrie Lowney (Zoetis)
Likely Webinar Attendees
Jim Zappia (3M)
Ed Carney (Dow Chemical)
Paul Jean (Dow Corning)
Kathy Plotzke (Dow Corning)
Yeyejide Adeleye (Unilever)
Paul Carmichael (Unilever)
Andy White (Unilever)
Hamner Staff Presenters
Melvin Andersen
Pergentino Balbuena
Sudin Bhattacharya
Michael Black
Harvey Clewell
Rebecca Clewell
Chad Deisenroth
Bo-wen Huang
Edward LeCluyse
Patrick McMullen
Michelle Miller
Salil Pendse
Bin Sun
Joe Trask
Barbara Wetmore
Miyoung Yoon
Qiang Zhang
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TT21C: Toxicity Pathways & Network Biology
Poster Abstracts
Poster Abstracts
1.
Safety assessment implications of differential TCDD dose responses across the
liver
MICHAEL BLACK
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent activator of signaling through the arylhydrocarbon
receptor (AhR) and causes liver cancer in rats. A significant amount of work has now explored the mode of
action for these tumors. Working together with collaborators from Dow Chemical, Hamner has investigated
gene expression patterns caused by treatment of rat primary hepatocytes with TCDD and gene expression
profiles from liver tissue from rats dosed with TCDD. We have also examined expression profiles from human
primary hepatocytes treated with TCDD. In the past year, we extended these studies by examining gene
expression and functional ontology responses for centrilobular (CL) and periportal (PP) hepatocytes from rats
exposed to 5 doses of TCDD (3, 22, 100, 300 and 1000 ng/kg/day). Cells were isolated by laser capture microdissection. Gene expression (average gene-based fold change) was greater in CL compared to PP for similar
doses although a heat map presentation showed that the gene expression pattern was fairly similar for the two
cell types. Enrichment for the highest dose group (equivalent to daily doses associated with carcinogenicity)
showed differential enrichment of pathways – cell cycle regulation and WNT/TGF signaling (PP) and cell
adhesion, immune response and ECM remodeling (CL). A key growth factor involved in hepatocyte precursor
differentiation (Hnf6) is down-regulated at the highest dose in both cell types. Hnf6 was not changed in the
human primary hepatocytes treated with TCDD. Safety assessments should primarily focus on the high dose
behavior both for understanding proliferative responses arising in PP hepatocytes and to ascertain if key
regulatory transcription factors and cell cycle pathways are also affected by TCDD in human hepatocytes.
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Poster Abstracts
2.
Tools to examine differential responses in the liver: a spatial multicellular model of
AhR pathway responses
SUDIN BHATTACHARYA
Multi-scale spatial “virtual tissue” models provide a computational framework to link early initiating events in
adverse outcome pathways (AOP), e.g., receptor activation or tissue reactivity, to tissue-level phenotypic
outcomes, e.g., cell proliferation or stress pathway activation. We have developed a multicellular virtual tissue
model of the liver lobule to predict biological responses to dioxin (TCDD)-induced aryl hydrocarbon receptor
(AhR) activation. A decreasing linear gradient of Wnt signaling was applied across the lobule model from the
centrilobular (CL) to the periportal (PP) regions. Ultrasensitive interactions reproduced sharply non-linear
expression of APC and beta-catenin across the lobule, in accordance with experimental observations. Bin the
model, beta-catenin in turn induces preferential centrilobular expression of AhR and activation of downstream
batteries of genes, including cytochromes 1A1, 1A2 and 1B1. Our multi-scale model recapitulated observed
progression of gene induction patterns from CL to pan-lobular with increasing dose of TCDD. At higher doses
of TCDD, Wnt-beta-catenin signaling would be more strongly activated in precursor cell populations proximal
to the PP end of the lobule, leading to induction of genes associated with proliferation and cell-cycling, as seen
in our laser capture microdissection study with TCDD. Overall, this multi-scale computational model
reproduced experimentally observed dose-response behaviors for TCDD-induced AhR activation and, with
continuing refinement, could support modeling of key events, i.e., proliferation, in the AhR AOP.
3.
Developing an in vitro high-content imaging assay for a measure of adversity proliferation in mouse hepatocytes
CHAD DEISENROTH
The two-year rodent bioassay is the standard metric for determination of carcinogenicity from exposure to
synthetic or natural chemicals. This assay is labor- and cost-intensive with limited throughput for assessing
hepatocarcinogenesis of novel chemicals with different modes of action. Transition to an in vitro model has
been hampered by challenges with reproducing proliferative responses in cultured primary hepatocytes. Using
an array of recombinant growth factors, cytokines, and model CAR activators, an effort was made to develop an
in vitro high-content imaging based assay for quantitative assessment of nascent DNA synthesis in primary
cultures of male CD-1 mouse hepatocytes. Detection of DNA synthesis was performed using click chemistry
labeling of the nucleoside analog 5-ethynyl-2′-deoxyuridine (EdU). Optimization of the DNA labeling index
revealed time- and seeding density- dependent effects to growth factor induced DNA synthesis. The assay was
multiplexed to enable direct quantitation of DNA synthesis, cytotoxicity, and cell count endpoints. Hepatocytes
were responsive to the CYP induction properties of known CAR activators TCPOBOP and phenobarbital. Using
an optimized defined medium cocktail, the EdU labeling response was enhanced in TCPOBOP treated
hepatocytes in a concentration-dependent manner. Additional reference compounds Oxazepam and CITCO
exhibit expected species-dependent outcomes on DNA synthesis. Our in vitro model of hepatocyte DNA
synthesis should aid in understanding proliferative responses to chemicals with CAR, and other nuclear receptor,
modes of action and their relevance for human safety assessments.
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Poster Abstracts
4.
Improving the Assay System for Oxidative Stress Responses: Comparison of
Human HaCaT and Primary Epidermal Keratinocyte Cells
BOWEN HUANG
Oxidative stress is the balance between production of reactive oxygen species (ROS) and the ability of the
system to cope with ROS. Nuclear factor E2-related factor 2 (Nrf2) plays a critical role in ROS defense by
binding the antioxidant response element (ARE) and initiating transcription of antioxidant genes. We set out to
measure the doses of chemicals inducing oxidative stress progress through three stages—sub-threshold,
adaptive, and adverse cellular responses to assess regions of safety for prototype oxidative stressors (curcumin,
hydrogen peroxide; H2O2). In HaCaT keratinocyte cells, markers of ROS and adaptive antioxidant responses
(i.e., Nrf2 accumulation, ARE transactivation, and expression of antioxidant genes) were induced by H2O2 and
curcumin, but no significant oxidative damage occurred to macromolecules. In contrast, both compounds
induced strong increases in protein nitration and carbonylation at non-cytotoxic concentrations in the primary
HEKn keratinocyte cells showing that HEKn cells are more sensitive to oxidative damage than HaCaT cells. In
an effort to advance the transition to in vitro toxicity testing described in the National Academies of Sciences
report, “Toxicity Testing in the 21st Century (TT21C): A Vision and A Strategy”, these time- and dose-response
data now support development of a computational model for oxidative stress response that will provide proof of
concept in vitro based safety assessments for oxidative stress-inducing compounds.
5.
Posttranslational control of adaptive cellular stress responses is an important
consideration in toxicity testing and risk assessment
QIANG ZHANG
Posttranslational control of stress response pathway activities serves to maintain homeostasis and efficiently
respond to low-level stressors. Using simple computational models, we show that posttranslational control has
two roles: (i) quickly managing small, transient stresses, and (ii) protecting the negative feedback transcriptional
network from oscillation. These pathways are at work in the posttranslational control pathways for oxidative
stress, heavy metal stress, hyperosmotic stress, DNA damage, heat shock, and hypoxia. Posttranslational
regulation of stress protein activities occurs by reversible covalent modifications (such as phosphorylation and
oxidation), competitive inhibition (as in glutathione synthesis), and protein structural changes caused by heat or
mechanical force. Posttranslational control, acting as a feedback or feedforward mechanism, also produces a
threshold against external perturbations. In this manner, post-translational regulation handles sub-threshold
levels of stressors. Supra-threshold stressor levels induce stress genes and other genetic program, e.g., those
regulating cell metabolism, proliferation and apoptosis. These higher-dose transcriptional alterations are likely
to be associated with adverse cellular outcomes on longer-term exposures. As cell-based in vitro assays become
a more significant focus for chemical testing anchored on perturbations of toxicity pathways, examination of
proteomic and metabolomic changes as a result of posttranslational control deserves more attention.
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The Hamner Institutes
TT21C: Toxicity Pathways & Network Biology
Poster Abstracts
6.
Understanding threshold processes in genotoxicity: Dose response modeling for
DNA repair center formation and resolution
SALIL PENDSE
The National Academies of Sciences 2007 report, “Toxicity Testing in the 21st Century: A Vision and A
Strategy” recommends toxicity testing using well-designed in-vitro assays to examine both adaptive and adverse
responses by using very broad dose ranges and coupling results from these assays with computational systems
biology and in vitro-in vivo extrapolations to complete human safety assessments. The current OECD
genotoxicity testing program includes in vitro micronucleus (MN) assays. The in vitro study will be more
specific, informative and meaningful for predictive or discovery toxicology if mechanistic consideration can be
linked to adverse outcomes. We created a computational model to study the mechanism of DNA repair center
formation and their resolution in response to double strand breaks (DSB). The model simulates the repair center
dynamics in response to neocarzinostatin, a DSB causing agent. Early computational model development
allowed us to gain an understanding of the processes affecting repair center formation and better understand the
conditions that can lead to thresholds for mutagenic chemicals. We are now extending the work to include MN
formation thereby linking model resul6s to existing toxicity data for chemicals. We also are looking at other
compounds that cause DSB to assess the scope of applicability of our computational model.
7.
Tools for assaying DNA repair at doses without mutagenic activity: looking at
double strand break repair kinetics in live human HT1080 cells
BIN SUN
In cancer risk assessment, USEPA and other agencies use default linear dose-response approaches. Our work
with DNA repair center (DRC) formation demonstrates that this default risk assessment may be incorrect for
most DNA-damaging compounds. We studied neocarzinostatin (NCS) and etoposide (ETP). NCS mimics
radiation damage and ETP inhibits DNA-repair processes. NCS has a threshold-like dose response behavior,
while ETP is more low-dose linear for MN formation. We have now examined the dose and time response for
individual p53BP1 DNA repair center (DRCs) in live human fibrosarcoma HT1080 cells to better understand
the molecular basis of these dose-response relationships. With NCS, DRCs peaked at 2h with no further DRC
formation after 2h. At low dose of NCS, DRCs were more efficiently resolved than after exposure to higher
doses. With ETP the initial DRC formation reached highest levels after 3hr and very few DRCs were resolved
between 3 and 12h regardless of dose. With ETP, the differential DRC time-courses compared to NCS appear to
be due to stability of ETP in the assay system and its mode-of-action in inhibiting DNA-repair. Our results
provide strong evidence in support of threshold dose response for MN formation. The linear behavior with ETP
strongly supports the requirement for efficient repair in producing these thresholds.
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TT21C: Toxicity Pathways & Network Biology
Poster Abstracts
8.
Confirming appropriateness of testing in cell lines: Characterization of estrogenic
responses in human endometrial primary epithelial and Ishikawa cells
PERGENTINO BALBUENA
Development of fit-for-purpose in vitro assays to study cellular signaling and functional response is essential for
characterizing toxicity pathway structure and measuring adverse perturbations by exogenous chemicals. To
identify the most appropriate model for evaluating effects of estrogenic compounds on uterine cells, we
compared the ability of two in vitro models to recapitulate the proliferative response to ethinyl estradiol (EE)
observed in vivo. Human endometrial epithelial primary cells (HEEC) and a human uterine epithelial
adenocarcinoma cell line (Ishikawa, IK) were examined. Proliferation in response to EE was observed in both in
vitro models with the MTS assay. The primary uterine cells (HEECs) demonstrated the greater induction, with a
maximum of 1.8-fold change and a lowest effective concentration of 10 pM. The cancer cell line showed ~1.2fold induction. Expressions of five estrogen receptors (ERs) that modulate proliferative signaling were evaluated
to help identify the basis of the differential response. The IK cells express full-length estrogen receptor ERα
(ERα66), its isoforms ERα36 and ERα46, the estrogen binding G-coupled protein receptor (GPER) and ERβ
proteins. Surprisingly, while ERα36, ERβ and GPER mRNA and proteins were observed in HEECs. These cells
had very low levels of ERα66 and ERα46 mRNA. Low levels of ERα66 protein in HEECs show that estrogen
mediated-proliferation is not solely dependent on full-length ERα66. Other non-ERα66 pathways may be more
important for cell proliferation in the uterus. Current high throughput screening efforts focus solely on ERα66
activation in breast cancer cell lines to predict estrogenic responses. Our results argue for more attention to the
criteria for designing fit-for-purpose assays for estrogenic compounds.
9.
Estrogen-mediated proliferation requires contributions from multiple receptors –
not simply ERα66
MICHELLE MILLER
Traditional testing protocols for endocrine disruptor screening are changing as the field moves towards fit-forpurpose in vitro assays to predict regions of safety. To replace current in vivo assays, we need a better
understanding of the key events in steroid receptor signaling. We are using human endometrial Ishikawa (IK)
cells that demonstrate phenotypic responses to examine the role of estrogen receptor (ER) isoforms in estrogenmediated proliferation. IK cells express all five key ERs: ERα66, ERα46, ERα36, ERβ, and GPER. Using
selective ER agonists, we have evaluated the role of each of the ER receptors in proliferation. Compounds that
selectively bind ERβ (DPN) and GPER (G1) did not induce proliferation. The ERα-specific ligand (PPT)
induced robust proliferation, indicating ERα66 is the main receptor mediating proliferation. However, PPT may
bind all three ERα isoforms, and we needed to determine if individual isoforms had unique contributions to
uterine cell proliferation. We designed systems to overexpress each of the isoforms in the IK cells and used
high-content imaging to confirm expression of the proteins in the cells. Increasing levels of ERα66 or ERα36
increased baseline proliferation in IK cells; ERα66 further increased both sensitivity and proliferative response
to ethinyl estradiol (EE). ERα46 overexpression, however, reduced EE-induced proliferation. Overall, ERα66
and ERα36 initiate proliferative signaling while ERα46 serves to reduce proliferative pathways. Our studies
emphasize the importance of multiple receptors for E2-mediated proliferation and highlight the need for EDCrelated assays that focus on more processes than ERα66 binding or E2-mediated transactivation.
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TT21C: Toxicity Pathways & Network Biology
Poster Abstracts
10. PPARα activation in rats: differential in vivo and in vitro responses influence assay
design
PATRICK MCMULLEN
For about 30% of compounds with reference doses, liver toxicity serves as the endpoint used for standard
setting. There is great uncertainty about the human relevance of these liver responses, e.g., increases in liver
weight, cell proliferation, and even non-genotoxic carcinogenicity. A major challenge in chemical risk
assessment is the degree to which liver responses in rodents are relevant for human safety. These differences
become even more problematic with new in vitro assays based on human cells/cell lines used to align with
legacy studies from rodents. Our work with PPARα signaling focuses on developing in vitro fit-for-purpose
assays that examine appropriate endpoints in rodent cells and tools to determine relevance for similar assays
using human cells. In vivo, sustained PPARα activation leads to tumors in rats, but apparently not in humans. To
better understand this mechanism, we exposed primary rat hepatocytes to a PPARα-selective agonist GW7647
over a range of concentrations (0.001–10μM) and did an in vivo study where with male rats exposed to
GW7647. Each included microarray evaluations. In vivo, higher doses gave cell proliferation with gene markers
for cell cycle. In vitro corresponding signals were seen for wound healing/cytoskeletal reorganization but not
cell cycle. These pathways were absent in human cells. Ancillary studies indicated that a rat specific network
associated with downregulation of key transcription factors (Hnf6 and Ets1) may be causal for “priming”
hepatocytes for proliferation (induction of cytoskeletal reorganization). Our results show that in vitro assays
with rat hepatocytes are misleading for estimating human regions of safety for cancer endpoints. Conversely,
with human cells, a focus on regions of safety might be based on activation of pathways for altered metabolic
processes.
11. TT21C at The Hamner: Creating new in vitro tools for quantitative safety
assessment
THE HAMNER TT21C TEAM
The goal of the Toxicity Pathways & Network Biology program at The Hamner is to develop in vitro toxicity
pathway testing platforms that can be used by industry partners and regulators for chemical risk/safety
assessment decision-making. We have developed a portfolio of research toxicity pathway-oriented projects
designed for understanding how dose-dependent processes lead to adverse responses, focusing on issues of
importance for our sponsoring organizations. Here, we highlight Hamner team progress in developing in vitro
fit-for-purpose assays to support chemical safety assessment and highlight how different aspects of our research
program fit into this pipeline.
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The Hamner Institutes
TT21C: Toxicity Pathways & Network Biology
Poster Abstracts
12. Developing approaches to assess differential regional hepatocyte responses in
humans
EDWARD L. LECLUYSE
Assessing liver responses to chemicals relies on fit-for-purpose assays using primary hepatocytes from rodents
or humans. Our projects with AhR and PPAR signaling in rodent liver indicate that hepatocytes across the liver
acinus respond very differently. Centrilobular hepatocytes (zone 3) are more sensitive with responses at lower
exposures. However, responses that are more aligned with potential adversity, such as cell cycle and
proliferation markers, appear to preferentially arise from activation of the periportal hepatocytes (zone1) at
higher doses. We have studied these differential responses by taking livers from rats after dosing and isolating
specific hepatocyte populations by laser capture microdissection. We currently lack the necessary tools to look
at preferential regional responses in human livers. The goal of this project is to develop methods to enrich CL
and PP hepatocytes from human tissues and assess differential responses to various chemical. We have devised
a unique method to enrich for several subpopulations of liver cells from either rat or human liver tissue using
counterflow elutriation centrifugation. With rats, one fraction contains predominantly small diploid cells that
express markers of zone 1 hepatocytes and relatively low levels of cytochrome P450 (CYP) activities. Another
fraction (F6) has many more large polyploid cells that express markers of zone 3 hepatocytes with relatively
high CYP activity. The purity of the preparation can be assessed by comparing gene expression profiles in the
elutriated fractions with profiles obtained by LCM form untreated liver preparations. In our initial studies
examining the proliferative potential of these cell fractions it appears that F4 cells proliferate under specified
growth conditions in vitro, whereas F6 cells do not. These enriched cell fractions potentially represent important
new tools to assess species differences in nuclear receptor biology and to guide development of liver stem cell
products that have more specificity in terms of the type of hepatocyte generated from various differentiation
protocols.
13. Improving quantitative in vitro to in vivo extrapolation using a liver bioreactor to
assess metabolism
MIYOUNG YOON
The goal of this project is to provide an in vitro system that generates in vivo-like metabolic profiles. This type
of work is important because metabolism is a major determinant for in vitro to in vivo extrapolation with results
from vitro-based fit-for-purpose assays. In the intact organism, the liver is the major contributor to metabolism.
Currently, however, it is difficult to evaluate hepatic metabolism of compounds due to limitations in hepatocyte
culture methods. To improve their predictivity, Hamner is developing long-term hepatocyte cultures that retain
metabolic capacity for many weeks. We are now evaluating both commercial and in-house developed dynamic
flow devices (bioreactors) loaded with hepatocytes and held for up to 30 days under different culture conditions.
Metabolic competence of the cells was measured by looking at 7-ethoxycoumarin (7-EC) metabolism.
Metabolic clearances and metabolite profiles of two case compounds, 7EC and acetaminophen, were then
determined and compared to profiles observed in vivo. To assist these comparisons, we have also developed a
kinetic model of the bioreactor. These 3D fluid dynamic modeling tools have assisted in the optimal design of
the culture system. Although species- and exposure scenario-specific metabolic profiles were reproduced
qualitatively, kinetics profiles were still significantly influenced by system-specific factors. The studies to date
show that careful considerations of both the kinetics of the compound and metabolites in the bioreactor and the
intrinsic metabolic clearances of the cells are critical for proper in vitro to in vivo extrapolation. The most
promising candidate for long-term 3D culture for metabolism appears to be hepatocytes cultured within alginate
beads. (Supported by ACC-LRI).
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The Hamner Institutes
TT21C: Toxicity Pathways & Network Biology
Poster Abstracts
14. IVIVE modeling and high-throughput tools to advance toxicity testing and risk
assessment
BARBARA A. WETMORE
Since its release, “Toxicity Testing in the 21st Century” has generated a great deal of interest and spin-off
research to assess the utility of high-throughput and in vitro screening for hazard identification. While a shift
away from traditional methods reliant on high dose in vivo testing is overdue, the potential application of in vitro
bioactivity screens for assessing regions of safety is limited without characterizing exposure potential for the test
compounds and the manner in which exposure determines tissue dosimetry in a population.
Recent and ongoing research efforts at The Hamner aim to develop and refine strategies that can be incorporated
with Tox21/ToxCast or other initiatives geared toward NexGen risk assessment. Some of these research efforts
include:
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Incorporation of chemical dosimetry with in vitro assay data, which allows an estimation of external
dose required to achieve internal concentrations at which assay activity is observed. These dosimetryadjusted oral equivalents can then be directly compared to human exposure estimates, enabling the
consideration of dosimetry and exposure with HTS data in assessing risk relevancy of a chemical;
Quantifying pharmacokinetic variability across different life stages and ethnic populations to inform the
range of population variability anticipated that may influence human health effects following chemical
exposure; and
Assessing a high-throughput, in vitro genomic platform that may better inform regarding chemical mode
of action than currently employed in vitro testing strategies
This poster highlights Hamner activities in these three key areas. (Supported by ACC-LRI).
15. Establishment of sensitive, quantitative and real-time cellular assays for
assessment and screening of modulators of endogenous androgen receptor signaling
pathways
PETER BENT
Cell based assays for detection of androgen receptor (AR) modulators were developed using real time
impedance technology. Two androgen responsive human prostate cancer cell lines: 22Rv1 and LNCaP, were
used for the study. Stimulation of these cells with androgen modulators lead to alterations in cell number and
cell adhesion, which can be detected by gold microelectrodes embedded in the bottom of the well of specialized
microelectronic plates. The time-dependent cellular kinetic response profiles were different in 22Rv1 and
LNCaP cells, indicating distinctive endogenous androgen signaling pathways in these two cell lines. Both cell
types exhibited low to high picomolar EC50 values, indicating that very high sensitivity to androgen receptor
stimulation. The specificities of the assays for AR activity were established using "pure" AR antagonists, such
as bicalutamide and nilutamide, and chemicals known with anti-androgen side effect, such as vinclozolin. More
interestingly, when LNCaP cells were starved, the kinetic response profile to AR agonist was changed,
reflecting altered native androgen response pathways in response to changes in growth condition. In addition,
under this condition, consistent with reported effect of the T877A mutation in LNCaP AR, nilutamide and
vinclozolin displayed AR agonist rather than AR antagonist effects in the real time cellular assay. The data
suggests that the impedance based real time cellular assay system has the capacity to sensitively, selectively and
quantitatively detect endogenous AR responses. The information can be useful to understand endogenous AR
signaling pathways, and to develop new AR ligands for therapeutic applications.
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