The Toxicologist - Society of Toxicology

1900 GENERATION OF PRECISION CUT LIVER SLICES
FROM PINCH BIOPSY SECTIONS.
K. Kowalkowski, A. Lisowski, W. R. Buck, E. Blomme and Y. Yang. Abbott
Laboratories, Abbott Park, IL.
Precision-cut tissue slices retain the multicellular, structural and functional features
of tissues and offers a more relevant approach to interrogate toxicity compared to
cell-based in vitro techniques. This study examined the feasibility of generating viable
tissue slices from biopsy-derived liver. In separate experiments, liver sections
were removed from dogs and monkeys. One set of sections was removed using a traditional
8-mm coring tool, and the other set was removed using a pinch biopsy forceps,
to replicate in vivo collection. For the traditional cores, slicing proceeded
using standard methods. To facilitate slicing, the much smaller biopsy sections were
first embedded in 2% agar to create 8-mm diameter plugs. Slices were cultured for
48 hours with 100 μM Rotenone as positive control for liver toxicant, with samples
taken at 24 hours as intermediate time point. Supernatants and tissue homogenates
(n=3) were analyzed for liver enzymes (ALT, AST and LDH) as endpoints for toxic
effects. Slices were processed for histological evaluation. All vehicle-treated slices
showed similar viability and low release for all three liver enzymes. In rotenonetreated
slices there were differences between species and time points. In dog liver,
histological examination showed rotenone-induced time-dependent hepatocellular
necrosis correlating with significant release of enzymes. The severity was comparable
in the biopsy and whole slices. In the monkey, there were no significant increases
of enzymes at the 24 hour time point, but after 48 hours significant levels of
all three enzymes were released from the traditional slices. For the biopsy slices, the
only significant change was in the LDH levels. These data demonstrate the feasibility
of using biopsy sections as a source of liver slices, which would enable toxicological
evaluation without sacrificing animals, and justify further exploration and refinement
of this technique.
1901 A HUMAN BREATHING LUNG-ON-A-CHIP FOR DRUG
SCREENING AND NANOTOXICOLOGY
APPLICATIONS.
D. Huh 1, 2 , B. D. Matthews 2 , A. Mammoto 2 , M. Montoya 1 , H. Hsin 2 and D.
E. Ingber 1, 2, 3 . 1 Wyss Institute for Biologically Inspired Engineering at Harvard
University, Boston, MA, 2 Children’s Hospital Boston and Harvard Medical School,
Boston, MA and 3 School of Engineering and Applied Sciences, Harvard University,
Cambridge, MA. Sponsor: A. Bahinski.
A major problem slowing the development and regulatory approval of new and
safer medical products is the lack of experimental in vitro model systems that can
replace costly and time-consuming animal studies by predicting drug efficacy and
toxicity in man. Here we describe a biomimetic microsystem that reconstitutes the
critical functional alveolar-capillary interface of the human lung and exposes it to
cyclic mechanical strain and fluid dynamic forces that mimic breathing and blood
flow. This microdevice reproduces complex integrated organ-level responses to bacteria
and inflammatory cytokines introduced into the alveolar space by inducing expression
of intercellular adhesion molecule-1 (ICAM-1) on the microvascular endothelium
surface, adhesion of circulating blood-borne neutrophils, their
transmigration across the capillary-alveolar interface, and phagocytosis of the infectious
pathogens, which can be visualized using real-time, high-resolution microscopy.
Using this approach, we developed novel nanotoxicology models and revealed
that physiological cyclic mechanical strain greatly accentuates toxic and
inflammatory responses of the lung to silica nanoparticles by promoting rapid release
of reactive oxygen species by alveolar epithelial cells and upregulating endothelial
ICAM-1 expression. Mechanical strain also enhances nanoparticle uptake
by the epithelial cells and stimulates their transport into the underlying microvasculature.
Importantly, similar effects of physiological breathing on nanoparticle absorption
were observed in whole lung using a mouse lung ventilation-perfusion
model. This mechanically active biomimetic microsystem represents valuable new
model systems for in vitro analysis of various physiological functions and disease
processes, in addition to providing low-cost alternatives to animal and clinical studies
for drug screening and toxicology applications.
1902 IN VITRO PREDICTION OF INJECTION SITE
REACTIONS USING L6 RAT SKELETAL MUSCLE CELLS.
J. A. Willy 1 , N. Schulte 2 , E. Kreklau 1 , J. Walgren 1 , A. Stauber 1 , J. L. Stevens 1
and T. K. Baker 1 . 1 Toxicology, Eli Lilly and Co., Indianapolis, IN and
2 Preformulation, Eli Lilly and Co., Indianapolis, IN.
Injection site reactions (ISRs) are commonly encountered in the development of
parenteral drugs. Severe ISRs can lead to preclinical and clinical dose limiting toxicities.
We developed an in vitro ISR screen to rank compounds for irritation risk, as
a surrogate for ISRs, during early preclinical development. Reference compounds
that were either known to induce ISRs or were non-irritating in vivo were used to
validate this method. L6 rat myoblasts were allowed to form a confluent monolayer
in 96 well plates followed by compound treatment for one hour at eight concentrations.
Change in membrane integrity was measured using calcein-AM. Cells treated
with non-irritants or non-irritating concentrations of compounds take up calcein-
AM and convert it to fluorescent calcein which is detected using laser scanning cytometry.
When membrane damage occurs, the fluorescent signal decreases in a concentration-dependent
manner and comparison of concentration response curves
allows for ranking of compounds. Using this assay, reference compounds were
ranked in the following order (from highest to lowest irritation potential): diethylstilbestrol
> mitoxantrone > mitomycin > doxorubicin > dacarbazine > vinblastine
> cis-platinum > cyclophosphamide > sumatriptan succinate > metoprolol ≥
atenolol ≥ vasopressin ≥ ranitidine HCl. This rank order correlates with in vivo ISR
potential for these reference compounds and, thus, demonstrates the usefulness of
this assay in identifying potential irritants. While compound mediated ISR is a critical
screening output for this model, it also has utility in understanding the role of
dosing formulations in ISRs and the impact of formulation additives on shifting
the ISR potential.
1903 HEPARG CELL MODEL IN HIGH-CONTENT
SCREENING ASSAYS GIVES MECHANISTIC INSIGHT
INTO METABOLISM-MEDIATED HEPATOTOXICITY.
J. Mein 1, 3 , P. Walker 3 , M. Jacewicz 1, 3 , R. Annand 1, 3 , D. Steen 2 , C. Chesne 2 ,
H. Gill 2 and K. Tsaioun 1, 3 . 1 Apredica, Watertown, MA, 2 Biopredic, Rennes, France
and 3 Cyprotex, Macclesfield, United Kingdom.
Drug-induced liver injury (DILI) is the most frequent reason for the withdrawal of
an approved drug from the market, and it also accounts for up to 50% of cases of
acute liver failure. Assays detecting mechanisms of potential adverse drug reactions
(ADRs) allow early de-risking of drug discovery programs. High Content Screening
(HCS) allows multiple mechanistic observations to be made simultaneously in a
single cell; here we measure mitochondrial membrane potential, cytochrome C release,
membrane permeability and cell loss. This multi-parameter assay gives mechanistic
insight into the toxic insult on a cell. By conducting this assay in two human
hepatocyte-derived cell lines, a further insight into the mechanism of DILI is provided,
by identifying if the parent compound or reactive metabolites of the parent
compound are potential hepatotoxicants. HepG2 cells are used as a surrogate
model for liver toxicity (parent compound), whereas HepaRG cells are used to determine
whether metabolites may be responsible for cell toxicity. Differentiated
HepaRG is a metabolically competent cell line that displays many hepatocyte-like
functions, including human CYP expression. HepaRG cells are preferred to primary
human hepatocytes, which are hampered by the irregular availability, donor
variability and scarcity of donors. Both HepG2 and HepaRG cells were treated with
test compounds and indicators of cell health were measured with HCS instrument.
Diclofenac, cyclophosphamide, and acetaminophen were chosen as compounds
known to form reactive metabolites. HepaRG cells are found to be more sensitive at
detecting the toxicity to the compounds with reported metabolism-based mechanisms
of toxicities. This assay will allow the identification of compounds with potential
ADRs and DILI, and identify compounds with metabolism-mediated toxicity
risk
1904 A NOVEL RESAZURIN/RESORUFIN CELLULAR ASSAY
TO DETECT AND CHARACTERIZE REACTIVE ACYL
GLUCURONIDES.
M. McMillian, J. Sasaki, M. Singer, F. Xu and H. Lim. Johnson & Johnson,
Raritan, NJ.
Glucuronidation generally inactivates metabolites, making them more soluble and
easy to eliminate. However, acyl glucuronides (AG) of some drugs are highly reactive,
and may contribute to idiosyncratic reactions; NSAIDs provide many examples.
Increased reduction of the redox dye resazurin to the more fluorescent resorufin
was found to reflect reactive AG formation from many added parent
compounds in hepatocyte and HepG2 cultures. The rank order of responses of
compounds tested in this cellular assay at equimolar concentrations was somewhat
different than previously reported following AG migration on glucuronidated compounds
in KRH buffer. Responses ranged from very robust, such as diflunisal,
flufenamic acid, and ethacrynic acid, to moderately robust, such as diclofenac, mycophenolic
acid and flurbiprofen, to moderate, such as zomepirac and tolmetin, to
mild or inactive, such as furosemide, fenofibrate, and valproic acid. Quantification
of diflunisal and zomepirac AGs showed that more zomepirac AG was formed than
SOT 2011 ANNUAL MEETING 407