The Toxicologist - Society of Toxicology

serum-free media. At 24 hrs, the greatest concentrations of lindane were in tissues
with low serum concentrations and the lowest levels of lindane were found in tissues
in 100% FCS. Taken together, these results suggest that at low chemical concentrations
high serum levels can delay the entry of lindane into tissue in vitro. In
systems with high metabolic capacity this limited availability would likely extend
the in vitro half-life of a compound. (This abstract does not necessarily reflect U.S.
EPA policy).
2563 AN IN VITRO MODEL FOR THE STUDY OF
BACTERIAL COLONIZATION OF HUMAN
EXTRAPLACENTAL MEMBRANES: ROLE OF
ANTIMICROBIAL PEPTIDES.
E. Boldenow 1 , S. Jones 1 , C. Xi 1 , R. Lieberman 2 and R. Loch-Caruso 1 .
1 Environmental Health Sciences, University of Michigan, Ann Arbor, MI and
2 Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI.
Preterm birth is a widely recognized, costly public health problem and is the leading
cause of infant mortality. Though the etiology of preterm birth is not fully understood,
preterm birth is associated with infection of the gestational compartment.
We developed an in vitro system for the coculture of bacteria and human extraplacental
gestational membranes, with the aim of using this system to study toxicant
modification of microbial infection of the membranes. We used Streptococcus
agalactiae (GBS) because this bacterium is commonly found in the vaginal tract and
untreated GBS infection has been associated with preterm birth. Gestational membranes
were affixed to Transwell frames (without synthetic membranes) in such a
manner that the full thickness gestational membranes provided a tissue barrier between
the Transwell culture compartments. The GBS was added to the decidual
side of the membranes in the Transwell culture, and the tissues were cocultured for
4, 8 and 24 hours with GBS. GBS colony forming units were determined for both
the media and the homogenized tissue. Recovered GBS colony counts decreased
over time suggesting that a robust innate immune response remained active in culture.
Antimicrobial peptide expression was identified in the gestational membranes
using immunohistochemistry (IHC). Human beta-defensin (HBD)-2 expression,
but not HBD-1, HBD-3, HBD-5, cathelicidin or elefin, was greater in the amnion
of GBS-treated membranes compared to controls, suggesting that HBD-2 expression
in the gestational membranes may be responsible for the killing of GBS over
time. This in vitro system has potential applications for future studies of toxicant
influences on bacterial infection of gestational membranes. Moreover, our results
suggest that toxicant inhibition of the antimicrobial peptide HBD-2 deserves further
study as a possible mechanism by which toxicants could increase susceptibility
to gestational compartment infection.
2564 EVALUATION OF AN ORAL CARE PRODUCT SAFETY
SCREENING PROGRAM UTILIZING THE IN VITRO
SKINETHIC HUMAN GINGIVAL EPITHELIUM (RHG)
AND ORAL BUCCAL (RHO) MODELS.
L. Wurzburger 1 , P. Kazmi 1 , T. Re 1 , A. Alonso 2 , B. Bertino 2 , N. Barnes 3 , A. de
Brugerolle de Fraissinette 2 , A. Hilberer 3 , H. Raabe 3 , N. Wilt 3 and V. Srinivasan 1 .
1 L’Oreal USA Products, Clark, NJ, 2 SkinEthic Laboratories, Nice, France and
3 Institute for in vitro Sciences, Inc., Gaithersburg, MD.
Assuring the safety of personal care products without testing in animals is a goal
common to many personal care products manufacturers, due to both ethical concerns
for animal welfare, as well as the limited relevancy of animal models to predict
human responses. Towards this goal, the cosmetics and personal care industry has
increasingly relied upon human cell-based 3–dimensional reconstructed tissues to
evaluate the safety of their product candidates in various target tissues. Accordingly,
we have developed a program for screening the potential irritancy of teeth whitening
products in oral mucosal tissues using commercially-available oral buccal
(SkinEthic RHO) and gingival (SkinEthic RHG) models. Four formulations containing
H 2 O 2 at various concentrations and one sodium bicarbonate were tested at
four exposure times to determine ET 50 values. Three irritancy endpoints were measured
after each exposure: viability using the MTT conversion assay, the amount of
IL-1α released from the tissues, and histological changes. Both models presented
the same rank order of the five materials, with increases in the ET 50 values correlating
with decreases in H 2 O 2 concentration. The formula containing sodium bicarbonate,
however, was non-toxic in both models. Histological analysis confirmed the
MTT results and provided evidence of the chemical impact upon cellular and tissue
morphology. IL-1α release did not appear to be as sensitive as the MTT assay at
shorter exposure times, although it may be useful to differentiate among formulations
predicted by the MTT assay to be of low irritation potential. Our results suggest
that the MTT viability and histology endpoints in 3–D human oral reconstructed
tissues can provide useful predictive information to support an oral care
products safety program.
2565 TRANSLATIONAL RESPONSE TO ARSENITE LEADS TO
P-BODY AND PAB1P-CONTAINING GRANULE
ASSEMBLY IN THE YEAST SACCHAROMYCES
CEREVISIAE.
V. Stribinskis, M. W. Gordon, J. P. Moore, S. R. Ellis and K. S. Ramos.
Biochemistry and Molecular Biology, University of Louisville, Louisville, KY.
Environmental stress of eukaryotic cells rapidly inhibits translation initiation and
causes shuttling of a subset of cytoplasmic mRNAs and proteins away from the
translational machinery into foci termed cytoplasmic processing bodies (PBs) and
stress granules (SGs). These cytoplasmic aggregates contain different subset of proteins
and often associate with each other, suggesting dynamic linkages between
mRNA storage and decay. In mammalian cells, cytoplasmic mRNAs redistribute
during arsenic stress to both PBs and SGs; however, the mechanisms and signaling
pathways that reprogram mRNA translation and decay remain obscure. Yeast has
been successfully used to unveil networks of transcriptional reprogramming of gene
expression in response to arsenite. Here we demonstrate that arsenite transiently inhibits
translation initiation at doses that affect neither cell viability nor growth.
This inhibition leads to rapid induction of PBs and Pab1p-containing granules
(mRNA poly(A)-binding protein). In the context of signaling, we show that arsenite
induces rapid phosphorylation of eIF2α, which requires Gcn2 kinase. All aforementioned
events are not completely abrogated, but rather delayed in cells lacking
Gcn2 kinase, suggesting that phospho-eIF2α-independent mechanisms of translational
control are also induced by arsenite. In contrast to the observed colocalization
of translation initiation factors eIF4E and eIF4G with Pab1p in yeast SGs during
glucose depletion, eIF4E and eIF4G were absent from Pab1p-containing
granules upon arsenite stress, suggesting that mRNA relocalization to cytoplasmic
granules can occur without factors that promote closed loop mRNP complexes.
Our observations share similarities with those in mammalian systems, thus suggesting
that the power of yeast genetics provides a good model to study how cells regulate
cytoplasmic mRNAs in response to arsenite stress.
2566 VALIDATION OF IN VITRO DIGESTION MODELS FOR
POLYCYCLIC AROMATIC HYDROCARBON
BIOACCESSIBILITY FROM SOIL USING THE IN VIVO
SWINE MODEL.
K. James 1 , R. E. Peters 1 , B. Laird 1 , W. Ma 2 , M. Wickstrom 3 , G. Stephenson 2
and S. D. Siciliano 4 . 1 Soil Science, University of Saskatchewan, Saskatoon, SK,
Canada, 2 Stantec Consulting Inc., Guelph, ON, Canada, 3 Toxicology Centre,
University of Saskatchewan, Saskatoon, SK, Canada and 4 Toxicology Group,
University of Saskatchewan, Saskatoon, SK, Canada. Sponsor: L. Weber.
Polycyclic aromatic hydrocarbons (PAHs) are a class of lipophilic organic contaminants
commonly found in soil. Particular PAH compounds have been identified as
known carcinogens. The total carcinogenic potential of PAHs can be expressed as
the total potency equivalency factor (PEF), which is based on the relative potency
of each compound to benzo(a)pyrene. PAH exposure from soil primary occurs
through incidental ingestion and in-vitro digestion models, such as the Simulator
of the Human Intestinal Microbial Ecosystem (SHIME) and Relative
Bioaccessibility Leaching Procedure (RBALP), can be used to estimate the bioaccessibility
of ingested environmental contaminants. Bioaccessibility estimates from
in-vitro models need to be validated using an in-vivo model for use in Human
Health Risk Assessment (HHRA). The chemical properties of PAHs indicate that
they will preferentially partition to lipophilic phases, therefore in-vitro models may
require the addition of a lipophilic membrane to act as a lipid sink for PAH absorption.
Using eight PAH contaminated soils we compare the release of the total
PEFs from the SHIME and RBALP with and without the addition of a lipophilic
membrane and then compare these results with the PEFs released from the swine
model. For both models, the SHIME and RBALP, the addition of a lipophilic
membrane significantly (p < 0.05) increases the release of PEFs from soil. However,
the release of PEFs from the SHIME model with the addition of a lipophilic membrane
is the only model that correlates to the PEFs released from the swine model.
The amount of PEFs released in the swine model shows a relationship with the individual
soil fugacity capacity, indicating that individual soil properties influence
the release of PEFs.
2567 IN VITRO EVALUATION OF AIRWAY TOXICITY USING
THE EPIAIRWAY ORGANOTYPIC IN VITRO HUMAN
AIRWAY MODEL.
G. R. Jackson, J. Bolmarcich, H. Kandarova, S. Letasiova, M. Klausner and P. J.
Hayden. MatTek Corp., Ashland, MA.
Recent REACH legislation has heightened the need for validated in vitro airway
toxicity models. However, in vitro determination of airway toxicity potential is
problematic since submerged monolayer cell cultures are not amenable to dosing of
SOT 2011 ANNUAL MEETING 549