The Toxicologist - Society of Toxicology

have now compared the IC50 values between human, dog, rat and mouse for
Imatinib, Erlotinib, Dasatinib, Sorafenib and Sunitinib. Bone marrow cells from
each of the species and KIs were mixed with ColonyGel methylcellulose-based
media containing appropriate species-specific cytokines and plated in 35 mm
dishes (n=3). CFU-GM were enumerated on days 7-14 (species dependent) and the
IC50 values determined for each drug for each species. IC50 values determined
using dog marrow compared well with human values (e.g.IC50 for Imatinib,
Dasatinib and Sorafenib were 2.1, 0.01 and 2.3 μM for dog and 2.5, 0.008 and 3.5
μM for human CFU-GM). In addition, the rank order of the 5 KIs was similar in
dog and human assays. In contrast, the IC50 values for the same drugs in rat and
mouse assays differed significantly (e.g. IC50 for Imatinib, Dasatinib and Sorafenib
were 25, 0.32 and 42 μM for rat and 67, 0.9 and 120 μM for mouse CFU-GM)
and the compounds did not rank in the same order. These data suggest that rat and
mouse derived IC50 values for KIs may not accurately reflect the relative toxicity of
newly designed molecules in this compound class.
1173 DEVELOPMENT AND CHARACTERIZATION OF
HUMAN HEPATOCYTES DERIVED FROM INDUCED
PLURIPOTENT STEM CELLS (IPSC): A NOVEL IN
VITRO MODEL SYSTEM FOR ASSESSING DRUG-
INDUCED HEPATOTOXICITY.
V. L. Ott, P. Fuhrken, J. Luebke-Wheeler, C. Kannemeier, B. Swanson, W.
Wang and E. Nuwaysir. Cellular Dynamics International, Madison, WI. Sponsor:
K. Kolaja.
Hepatotoxicity and drug-induced liver injury (DILI) are the most common reasons
for drug failure during development and market withdrawal of approved drugs.
Liver diseases associated with drug toxicity can be attributed, in large part, to the
lack of biologically relevant and predictive model systems. Current hepatocyte
model systems include primary human hepatocytes (PHH) harvested from cadavers,
immortalized cell lines and animal models. Each of these presents limitations
in functionality, reproducibility and/or availability. Human hepatocytes derived
from induced pluripotent stem cells (iPSCs), which are stem cells derived from
adult tissue, offer the potential to overcome these limitations. We have developed
human iPSC-derived hepatocytes that are >95% pure and exhibit characteristic hepatocyte
morphology, gene and protein expression (e.g. albumin, alpha-1-antitrypsin,
ASGR1, HNF family transcription factors). Human iPSC-derived hepatocytes
produce albumin protein at levels similar to PHH and ~7-fold greater than
HepG2 cells. In addition, >50% of the cells store glycogen and lipid, as demonstrated
by Periodic Acid Schiff and Oil Red staining, respectively. Human iPSC-derived
hepatocytes express Phase I and II metabolic enzymes (e.g. CYP1A2, 2B6,
2C8/9/19, 2D6, 3A4, UGT, ST and GST) at levels similar to PHH, and CYP3A4
activity is induced ≥3-fold in response to dexamethasone and rifampicin. Finally,
human iPSC-derived hepatocytes express key uptake and efflux transporters (e.g.
MDR-1/P-gp, MRP2, BSEP, BCRP, NTCP and OATPs) and exhibit formation of
bile canaliculi. The development of human iPSC-derived hepatocotyes that recapitulate
in vivo hepatocyte function will enable better predictivity of drug-induced
hepatotoxicity during the earlier pre-clinical stages of drug development, more successful
clinical trials, and the delivery of new drug therapieeis across a wide range of
diseases.
1174 BIOLOGICAL SURFACE ADSORPTION INDEX (BSAI)
FOR CHARACTERIZING CARBON NANOMATERIALS
WITH DIFFERENT SURFACE CHEMISTRIES IN
BIOLOGICAL SYSTEMS.
X. Xia, N. A. Monteiro-Riviere and J. E. Riviere. Center for Chemical Toxicology
Research and Pharmacokinetics, North Carolina State University, Raleigh, NC.
The behavior of nanomaterials in a biological or environmental system is governed
by the molecular interactions of their surface species with the biological or environmental
components. Quantitative assessment of the adsorption properties of nanomaterials
is a crucial step for developing predictive structure-activity relationship in
nanomedicine and risk assessment of nanomaterials. We have developed a biological
surface adsorption index (BSAI) approach to characterize the surface activity of
nanomaterials in biological systems. A set of small molecules having diverse physicochemical
properties was used as probe compounds. The adsorption coefficients (k)
of the probe compounds were obtained by measuring the quantities of the probe
compounds adsorbed on the surfaces of the nanomaterials and the equilibrium concentrations
of the probe compounds in the media. The log (k) values were scaled to
a set of solvation molecular descriptors of the probe compounds via multiple linear
regressions to provide a set of five nano-descriptors representing the contributions
of the five types of molecular interactions (hydrophobicity, hydrogen-bond acidity
and basicity, dipolarity/polarizability, and lone pair electrons). The nano-descriptors
for multi-walled carbon nanomaterials (MWCNT) with different surface
chemistries (unmodified, -OH and –COOH modified) and fullerenes were measured;
for example, the regression model obtained for MWCNT (-OH modified)
was log(k) = 0.77R + 2.55π - 0.14α - 2.36β + 4.90V; n=30, R 2 =0.89. The measured
nano-descriptors can be used to develop predictive structure-activity relationships
in nanomedicine and nanomaterial risk assessments. (Supported by US EPA
STAR Grant# R833328 and NIH R01 ES016138)
1175 CELLULAR UPTAKE MECHANISMS AND
CYTOTOXICITY OF QUANTUM DOT NANOPARTICLES
IN PORCINE DENDRITIC CELLS.
L. W. Zhang and N. A. Monteiro-Riviere. Center for Chemical Toxicology Research
and Pharmacokinetics, North Carolina State University, Raleigh, NC.
Quantum dots (QD) serve as excellent fluorescent probes and are novel tools used
in biomedical applications but the toxicity and mechanism of cellular uptake are
poorly understood. Peripheral blood mononuclear cells were isolated from porcine
blood by gradient centrifugation and monocytes were purified with anti-CD14 antibody
conjugated beads. Monocytes dosed with 0.05nM of QD655 coated with
carboxylic acid (QD655-COOH, 18nm) for 30min showed cellular uptake while
lymphocytes did not take up QD655-COOH. Monocytes were differentiated into
dendritic cells (DC) with granulocyte-macrophage colony stimulating factor and
IL-4. Cellular uptake of DC increased six-fold compared to monocytes when cells
were dosed with 2nM of QD655-COOH for 30min. QD655 coated with polyethylene
glycol (PEG) (45nm) or QD655 PEG-amine (20nm) did not show endocytic
uptake in monocytes or DC. QD655-COOH uptake in DC was saturated at 4-6h
and TEM showed QD in the cytoplasmic vacuoles of DC. Twelve endocytic inhibitors
were used to investigate the cellular uptake mechanisms of QD655-
COOH in DC. These results showed that cytochalasin D and chlorpromazine inhibited
QD uptake by 50%, demonstrating that cytoskeleton F-actin and clathrin
regulated the QD655-COOH endocytosis of DC. The cellular uptake was primarily
regulated by scavenger receptor and phospholipase C. Scavenger receptor and a
phospholipase c (U-73122) inhibitors blocked QD uptake. In addition, DC maturation
with lipopolysaccharide (LPS) caused an increase in QD655-COOH uptake
compared to DC without LPS. Viability assays including 96AQ, CCK-8,
alamarBlue and ApoTox exhibited minimal toxicity in DC dosed with QD655-
COOH at 24h. However, glutathione levels showed a significant decrease with
10nM of QD655-COOH. Lastly, QD655-COOH not only suppressed
CD80/CD86 expression on the surface of DC but decreased LPS induced
CD80/CD86 expression. These findings shed light on the cytotoxicity and mechanisms
of QD cellular uptake in DC.
1176 EVALUATION OF TOXICITY AND INFLAMMATION IN
THREE DIFFERENT HYDROXYLATED FULLERENES
(C 60 (OH) X ) IN HUMAN CELLS.
J. G. Saathoff 1 , X. Xia 1 , J. E. Riviere 1 , A. O. Inman 1 , A. R. Badireddy 2 , M. R.
Wiesner 2 and N. A. Monteiro-Riviere 1 . 1 Center for Chemical Toxicology Research
and Pharmacokinetics, North Carolina State University, Raleigh, NC and 2 Civil and
Environmental Engineering, Duke University, Durham, NC.
Carbon fullerenes (C 60 ) possess unique chemical properties and their interactions
with biomolecules have widespread applications in biomedical technologies.
Functionalization of C 60 with hydroxyl groups can increase the solubility and potential
for cellular interaction. There is much speculation as to the potential benefits
of the medical application of solubilized fullerenes (i.e. MRI contrast agents,
target drug delivery, etc.), and although industrial use and production continues to
increase the health and safety effects of hydroxylated C 60 within biological systems
is poorly understood. Conflicting reports regarding the cytotoxicity and inflammation
of functionalized C 60 exists. Hydroxylated fullerenes (C 60 (OH) x ) were synthesized
using the prepared phase transfer method. In an attempt to further elucidate
the potential for toxicity of functionalized C 60 , human epidermal keratinocytes
(HEK) were exposed to three different hydroxylated fullerenes. C 60 (OH) 20 ,
C 60 (OH) 24 , and C 60 (OH) 32 at concentrations ranging from 0.000544 to
42.5μg/ml for 24 and 48hr (n=12 wells/treatment) were exposed to HEK, and
alamarBlue (aB) viability assay and IL-8 cytokine analysis were conducted. A statistically
significant (p