The Toxicologist - Society of Toxicology

77 DEVELOPMENT OF EMBRYONIC STEM CELL-
DERIVED NEURONS FOR BOTULINUM NEUROTOXIN
RESEARCH AND DRUG DISCOVERY.
P. McNutt and M. Mesngon. USAMRICD, Gunpowder, MD. Sponsor: G.
Rockwood.
Identification of effective small molecule inhibitors of botulinum neurotoxins
(BoNT) have not been successful, in large part due to the lack of a neuromimetic
cell-based platform. We report that neurons differentiated from embryonic stem
cells are compatible with drug discovery methodologies and suitable for molecular
characterization of intoxication. Suspension-adapted ESCs reliably differentiate
into homogenous neuron cultures (ESNs) that express BoNT substrate proteins,
exhibit action potentials, construct synapses and undergo trans-synaptic signaling.
ESNs display characteristic synaptic architectures, and extracellular electrophysiology
using multielectrode arrays (MEAs) verifies the presence of action potentials
and network activity. Exposure of ESNs to BoNT/A and /E results in cleavage of
target SNARE proteins with EC50s of 0.8 and 67.4 pM, respectively. These sensitivities
are equivalent to primary fetal spinal cord neurons and two-to-three orders
of magnitude improved over alternative cell-based model systems, suggesting that
ESNs internalize and process BoNTs similar to primary neurons. Exposure of ESNs
to 10xEC50s of BoNT/A between 5-90 days after plating causes complete
SNAP25 cleavage and inhibition of neurotransmitter release. The ability to generate
post-mitotic neurons via cell culture allows application of novel research
methodologies to BoNT drug discovery. For example, we are using systems biology
approaches to identify novel therapeutic targets for BoNT research. Candidate targets
will be interrogated by multiple methods, including neuronal expression of genetically
encoded reporters and knock-down of endogenous gene expression. We
are determining differentiation conditions that enrich for cholinergic motor neurons
in order to generate neuromuscular junctions in vitro for increased physiologic
relevancy. Finally, we are evaluating the use of multielectrode arrays as a networkbased
BoNT drug discovery program. We will discuss the utility of ESNs as a cellbased
in vitro toxicology platform for BoNT therapeutics as well as for other neuron-specific
insults.
78 TCDD-INDUCED MODIFICATION OF
TRANSCRIPTION FACTOR SIGNALING AND
CARTILAGE DYSMORPHOGENESIS IN VIVO .
W. Dong 1, 2 and S. W. Kullman1 . 1Department of Environmental and Molecular
Toxicology, North Carolina State University, Raleigh, NC and 2College of Animal
Science and Technology, Inner Mongolia University for the Nationalities, Tongliao,
Inner Mongolia, China.
Mesenchymal stem cells (MSC) are highly regulated multipotent stem cells that
give rise to multiple cell and tissue types including cartilage and bone. Formation of
cartilage consists of a highly coordinated and orchestrated series of events involving:
commitment and differentiation of mesenchymal cells to chondrocytes, programmed
and structured maturation and eventual formation of bone surrounding
the perichondral structures. Our laboratory has identified a novel hypural cartilage
phenotype in medaka that serves as a prototypic focus of cartilage dysmorphogenesis
following 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure. Here we establish
the localization and differential gene expression for key regulatory factors associated
with MSC transition to differentiated cell types including: Transforming
Growth Factor Beta-2 (TGF- β 2), Sry-Related High Mobility Group Box
Containing Gene 9 (SOX9), Paired Box Gene 9 (Pax9) and Runt-Related
Transcription Factor 2 (Runx2). In vivo exposure of medaka embryos to TCDD results
in a dose-dependent decrease in transcription factor expression for all genes examined
at 10 day post fertilization (dpf) using both RT-PCR and/or in situ hybridization.
A marked reduction in Col2a1 protein expression, a marker of
chondrocyte differentiation, is additionally observed at 10 dpf in medaka hypural
cartilage consistent with an observed reduction in hypural cartilage deposition.
79 METHYL MERCURY-INDUCED PERTURBATION OF
NEURAL DIFFERENTIATION OF MURINE
EMBRYONIC STEM CELLS OVER TIME DESCRIBED BY
TRANSCRIPTOMICS.
P. T. Theunissen1, 2 , J. L. Pennings1 , J. C. Kleinjans2 , J. F. Robinson1 and A. H.
Piersma1, 3 . 1Laboratory for Health Protection Research, National Institute of Public
Health and the Environment (RIVM), Bilthoven, Netherlands, 2Department of
Health Risk Analysis and Toxicology, University of Maastricht, Maastricht,
Netherlands and 3Institute for Risk Assessment Sciences, Utrecht University, Utrecht,
Netherlands.
Current chemical hazard assessment for developmental toxicity requires high numbers
of experimental animals. Alternative developmental toxicity assays are highly
desirable to reduce animal use. For screening effects on neural development, we
16 SOT 2011 ANNUAL MEETING
previously developed a dynamic in vitro model which makes use of neural cell differentiation
of pluripotent murine embryonic stem cells (Reprod. Toxicol. 2010
29:383-92). To further characterize this neural differentiation model and to improve
detection of developmental toxicants, gene expression patterns within the
model were studied. A transcriptomics study was performed to study whole
genome expression changes during the first 7 days of the protocol. Statistical analysis
was performed using R. Pathway enrichment analysis of GO terms was performed
using DAVID and T-profiler. Analysis of gene expression using PCA
showed a time dependent track in unexposed controls, describing the process of
neural differentiation. This was confirmed for specific gene sets using pathway enrichment
analysis. They revealed downregulation of blastocyst and trophectoderm
related genes and upregulation of neural development related gene sets over time.
Furthermore, the developmental toxic effects of methyl mercury (MeHg) on neural
differentiation over time were assessed. MeHg was shown to induce deviation from
the predefined differentiation track. The compound inhibited proliferation related
pathways and induced neural related pathways over time. This system appears
promising for studying compound effects on neural differentiation in a mechanistic
approach.
80 VALIDATION OF THE CANDIDA ALBICANS DELAYED-
TYPE HYPERSENSITIVITY (DTH) MODEL IN THE
B6C3F1 MOUSE FOLLOWING EXPOSURE TO
AZATHIOPRINE (AZA), CYCLOPHOSPHAMIDE (CPS),
CYCLOSPORIN A (CSA), DEXAMETHASONE (DEX), OR
BENZO[E]PYRENE (B[E]P) FOR 28 DAYS.
M. J. Smith, C. E. McLoughlin, W. Auttachoat and K. L. White. Pharmacology
and Toxicology, Virginia Commonwealth University, Richmond, VA.
Although numerous models are in use to evaluate the immunotoxic effects of a
xenobiotic on cell-mediated immunity (CMI), no single holistic model for evaluating
such effects on the DTH response has gained widespread acceptance. The C.
albicans DTH model has been suggested recently as a more appropriate model for
evaluating effects on the DTH response in mice than models using either sheep erythrocytes
(sRBC) or keyhole limpet hemocyanin (KLH) as sensitizing antigens.
The purpose of these studies was to validate the C. albicans DTH model for its ability
to detect suppression (or the lack thereof) of CMI following exposure for 28
days to well-characterized immunosuppressive drugs having different mechanisms
of action. The compounds evaluated included AZA, CPS, CSA, DEX, and the
non-immunotoxic compound B[e]P. Results from initial studies demonstrated that
the C. albicans DTH model was able to detect suppression of CMI for each known
immunotoxicant, with statistically significant decreases in the DTH response observed
following exposure to AZA (at 30 mg/kg but not at 10 mg/kg), CPS (at 30
mg/kg but not at 10 mg/kg), CSA (at 1 mg/kg, the lowest dose evaluated), or DEX
(at 0.3 mg/kg but not 0.1 mg/kg). As expected, B[e]P exposure for 28 days at doses
up to 40 mg/kg did not suppress the DTH response. These results indicate that the
C. albicans DTH assay in the B6C3F1 mouse was capable of appropriately classifying
each test article as to its immunotoxic effects on CMI. Furthermore, these results
further validate the use of this model in immunotoxicity testing. Supported in
part by NIEHS Contract ES 05454.
81 A RETROSPECTIVE OF THE REGULATORY USE OF
THE LOCAL LYMPH NODE ASSAY FOR THE
NOTIFICATION OF NEW CHEMICALS IN EUROPE.
S. Casati, A. Angers-Loustau and L. Tosti. European Commission (Joint Research
Centre), Ispra, Italy. Sponsor: D. Basketter.
We have monitored the regulatory use of the LLNA for chemicals registration from
the time of its adoption as stand alone method at the OECD to 2008. For this, we
screened the New Chemicals Database (NCD) which was managed during this period
by the former European Chemicals Bureau (ECB) at the European
Commission Joint Research Centre (JRC). The NCD comprises chemicals notified
after 1981, where registered data have been derived according to regulatory standards,
including GLP and predominantly according to official test methods. The
database was searched to extract records for which the information for skin sensitisation
labelling was based on results derived with the LLNA. The details of these
records were extracted and pooled, for a total of 545 entries, and evaluated with regards
to the extent of use of the LLNA over time and countries, as well as analysing
the information derived on critical aspects of the procedure e.g strain and amount
of animals used, lymph node processing, solvent and doses selected, stimulation indices,
and assessing their level of compliance to the OECD 429. Additional information
on labelling was extracted, and its significance for the potential of the widespread
use of the reduced version of the LLNA is discussed on the poster.
Disclaimer: The views expressed in this poster are purely those of the authors, and
should not be regarded as an official position of the European Commission.