The Toxicologist - Society of Toxicology

1963 CHARACTERIZATION OF HEPATOTOXICITY
INDUCED BY 1-FURAN-2-YL-3-PYRIDIN-2-YL-
PROPENONE, A NEW ANTI-INFLAMMATORY AGENT.
H. W. Ha 1 , T. W. Jeon 2 , G. S. Ko 1 , J. W. Yoo 1 , S. K. Lee 1 , M. J. Kang 1 , E. S.
Lee 1 and T. Jeong 1 . 1 Pharmacy, Yeungnam University, Gyeongsan, Gyeongbuk,
Republic of Korea and 2 BioToxtech Incorporation, Ochang, Republic of Korea.
1-Furan-2-yl-3-pyridin-2-yl-propenone (FPP-3) has recently been synthesized as
an anti-inflammatory activity through the inhibition of the production of nitric
oxide. In the present study, adverse effects of FPP-3 on hepatic functions were determined
in female BALB/c mice. When mice were administered with FPP-3 at
125, 250 or 500 mg/kg for 7 consecutive days orally, FPP-3 significantly increased
absolute and relative weights of liver with a dose-dependent manner. In addition,
FPP-3 administration dramatically increased the hepatotoxicity parameters in
serum at 500 mg/kg, in association of hepatic necrosis. FPP-3 significantly induced
several phase I enzyme activities. To elucidate the possible mechanism(s) involved
in FPP-3 induced hepatotoxicity, we investigated the hepatic activities of free radical
generating and scavenging enzymes and the level of hepatic lipid peroxidation.
FPP-3 treatment significantly elevated the hepatic lipid peroxidation, measured as
the thiobarbituric acid-reactive substance, and the activity of superoxide dismutase.
Taken together, the present data indicated that reactive oxygen species might be involved
in FPP-3-induced hepatotoxicity.
1964 ALGINATE SPONGES AS PHYSIOLOGICALLY
RELEVANT CULTURE ENVIRONMENTS FOR PRIMARY
RAT HEPATOCYTES.
A. Sams, Z. Li, M. Gonzalez, J. Jackson, J. Hill and M. Powers. PSCS R&D,
Invitrogen Corporation, Frederick, MD.
We have developed a lyophilized porous alginate matrix (AlgiMatrix) to establish
a physiologically relevant environment that facilitates the formation of more in
vivo-like tissue structures. The addition of divalent cationic salts (e.g. calcium, barium)
to a sodium alginate solution produces a highly porous (>90%) and highly interconnected
biocompatible scaffold. Using multiple cell types we have shown that
the matrix pores (50-150μm) act as localized compartments in which cells are able
to interact with one another (because cells do not significantly interact with the alginate)
to form 3D multicellular spheroidal structures. The technology is of particular
interest in the field of liver biology; hepatocellular spheroids have been shown
to maintain expression of key hepatic markers. However, these spheroids are traditionally
somewhat cumbersome to culture and maintain. Phase contrast microscopy
reveals that spheroid formation is faster and more efficient in
AlgiMatrix cultures compared to other currently available platforms (e.g. lowbind
polystyrene). Live/dead staining and intracellular ATP levels confirm that
AlgiMatrix cultures of primary rat hepatocytes retain a high viability through at
least 14 days in culture. Cellular toxicity measurements, including intracellular reduced
glutathione levels and caspase-3 activity, demonstrate that AlgiMatrix
provides a more suitable culture environment for primary hepatocytes in comparison
to conventional sandwich culture. AlgiMatrix cultures of primary rat hepatocytes
demonstrate enhanced basal cytochrome P450 (e.g. CYP1A, 2B, 3A) activity
and gene expression compared to conventional sandwich culture. Collectively,
these results suggest that AlgiMatrix is a potentially useful predictive tool in
xenobiotic metabolism and toxicology research. We conclude that 3D cell culture
scaffolds such as AlgiMatrix serve as a foundation for the creation of more physiologically
relevant culture systems.
1965 CHARACTERIZATION OF AN IMMORTALIZED
MURINE KUPFFER CELL LINE FOR USE IN
TOXICOLOGICAL STUDIES.
Z. Wang, J. E. Klaunig, B. Hocevar and L. M. Kamendulis. Pharmacology &
Toxicolgy, Center for Environmental Health, Indiana University School of Medicine,
Indianapolis, IN.
Kupffer cells play a critical role in both liver physiology and the pathogenesis of various
liver diseases. Isolated primary Kupffer cells have a limited lifespan in culture,
and due to the relatively low number obtained, limit their study in vitro. Here, an
immortalized murine Kupffer cell line was established from transgenic mice that express
the thermolabile mutant tsA58 of the Simian virus 40 large T antigen under
the control of the H-2kb promoter. Primary Kupffer cells were obtained using a
three step procedure: liver perfusion, centrifugal elutriation, and sorting for F4/80+
cells. A subpopulation of cells (ImKC) was identified within the small-intermediate
population of sorted Kupffer cells that maintained stable expression of the F4/80
and the surface antigens CD11b, CD14 and CD80. ImKCs maintained proliferative
capacity at 37C and exhibited a doubling time of ~24 hours when cultured in
RPMI 1640 with 5% FBS. ImKC cells maintained a high capacity to phagocytose
FITC-latex beads, in response to lipopolysaccharide (LPS; 0.1-1μg/ml). In addition,
similar to primary Kupffer cells, LPS treatment of ImKCs caused upregulation
of TNFα (23-fold), IL-6 (28-fold), IL-1β (1459-fold), IL-10 (14-fold), inducible
nitric oxide synthase (iNOS) (11-fold) mRNA levels as measured by qRT-PCR.
Protein levels of TNFα were also increased 10-fold; and ROS and TLR4 expression
were enhanced 2-fold and 2.6- fold, respectively. Furthermore, LPS caused a
marked increase in mitogen activated protein kinase (MAPK) phospho- (ERK1/2,
JNK) and NFkB p50 with decreased IκBα as assessed by Western blot. Moreover,
ethanol-pretreatment sensitized ImKCs to LPS resulting in enhanced TNFα production
through p38 activation, a finding not seen in the RAW 246.7 macrophage
cell line. Collectively, the ImKC line retains critical characteristics of primary
Kupffer cells, and thus provide a useful cell line to assess the role of Kupffer cells in
liver injury and chronic diseases (Supported in part by NIH CA100908).
1966 IN VITRO 3-D LIVER CO-CULTURES FOR ADMET
ASSESSMENT OF NEW COMPOUNDS.
D. R. Applegate, L. New and B. A. Naughton. RegeneMed Inc., San Diego, CA.
Sponsor: R. Thomas.
Many pharmaceutical companies are screening toxicity of drug candidates earlier in
the discovery process to increase success in later stages of drug development, often
using primary hepatocytes as an in vitro model of in vivo liver function. However,
primary hepatocytes lose P450 activity and albumin synthesis within a few days of
culture, and thus better in vitro models are needed. Three-dimensional (3D) tissue
technology was developed that provides a more physiologically relevant in vitro
liver by culturing all liver cells on a 3D scaffold to form a tissue with long-term
function. These cultures maintain liver specific function in culture for months, including
liver-specific protein expression, extracellular matrix protein deposition, receptor
activity, cytochrome P450 metabolism, induction and inhibition, and biliary
clearance. These functions have been previously published to 3 months in
culture and are now verified to 6 months including incorporation of recent assay
methods including genomics, proteomics, metabolomics, 3D imaging through the
tissue depth, and multiplexed assays. These 3D liver co-cultures have been grown
from a variety of species including mouse, rat, dog, nonhuman primate and human
liver donors in multiwell plates from 1 to 96 well, with 384 and 1536 well formats
in development. The liver co-cultures and assay methods serve as animal alternatives
in accelerating drug discovery.
1967 PHENOBARBITAL INDUCES TRANSCRIPTIONAL AND
FUNCTIONAL CHANGES WITH TIME IN 3-D LIVER
CO-CULTURES CONSISTENT WITH
HEPATOCARCINOGENESIS.
R. Brennan 3 , L. New 1 , R. S. Thomas 2 , C. I. Pearson 1 , A. H. Roter 4 , B. A.
Naughton 1 and D. R. Applegate 1 . 1 Research, RegeneMed Inc., San Diego, CA,
2
Genomic Biology and Bioinformatics, The Hamner Institutes for Health Sciences,
Research Triangle Park, NC, 3 Research, GeneGo, Inc., Encinitas, CA and 4 Research,
Entelos, Inc., Foster City, CA.
Many non-genotoxic compounds are tested for carcinogenic potential using the
“gold stardard” two year rodent bioassays. Because of its high cost and lengthy time,
many chemicals of concern have not been tested. Moreover, while rodent carcinogens
are generally considered human carcinogens, there are exceptions. Less costly,
high throughput assays are needed. A three-dimensional (3D) in vitro organotypic
liver co-culture provides a physiologically relevant model of intact liver by culturing
all hepatic cells on a 3D scaffold. These co-cultures form a tissue that maintains
liver functions including cytochrome P450 induction for months. To test their potential
in carcinogenicity assessment, 3D co-cultures were exposed to
Phenobarbital, a non-genotoxic hepatocarcinogen, for three weeks. Cell proliferation
measured by tritiated thymidine uptake occurred in mouse, rat, and human
co-cultures. Microarray data showed upregulation of Cyp2B and Cyp3A transcripts
and other CAR-inducible transcripts within the first 24 hours of exposure, and
maintained for at least 3 days. Transcriptional profiles of cell cycle pathways indicated
cell cycle progression and DNA replication at early time points in male rats,
whereas by 18 days, cell cycle arrest and oxidative stress were observed. Remodeling
of the male 3D co-cultures by day 18 was indicated by up-regulation of genes associated
with embryonic development, nervous system development, cell differentiation,
and GPCR activity, and down-regulation of collagens, CAR-inducible genes,
and acute phase response genes. These functional and transcriptional studies indicate
that 3D liver mirrors known in vivo effects leading to tumor formation, such as
CAR activation, cell proliferation, and oxidative stress, providing a much-needed in
vitro model suited for HTS assessment of hepatocarcinogenic potential.
418 SOT 2010 ANNUAL MEETING