The Toxicologist - Society of Toxicology

forms mRNA expression levels were assessed by quantitative real-time PCR assay
(rtPCR). A time-dependent variation of the mRNA expression levels was observed.
The maximum expression was detected at 12 h. The observed effect was similar for
the four genes studied. B[a]P treatment had no effect on the viability of cells.
This is the first study to our knowledge where lymphocyte cells are used to evaluate
the PAHs effect on the AKRs mRNA expression.
2525 DATACHIP/METACHIP 2.0: AN IMPROVED CHIP
PLATFORM FOR 3D CELL-BASED TOXICOLOGY
ASSAYS.
M. Lee 1 , J. Ryan 1 , S. Brooks 1 , J. Gray 1 , D. Lee 2 , S. Jeong 2 , J. Yang 2 , B. Ku 2 ,
Y. Will 3 and S. Nadanaciva 3 . 1 Solidus Biosciences, Inc., San Francisco, CA, 2 Central
R & D Institute, Samsung Electro-mechanics Co., Suwon, Republic of Korea and
3 Compound Safety Prediction, Pfizer Inc., Groton, CT.
Several studies suggest that 3-D cell cultures are more likely than 2-D cell cultures
to reflect accurate physiological responses to cytotoxic compounds. Solidus
Biosciences has developed the DataChip/MetaChip platform that couples a 3-D
cell culture array with a complementary array of recombinant enzymes to identify
metabolism-induced toxicity. The IC50 values of many compounds not only depend
on the morphology of the cultured cells, but also vary with culture conditions
and the cell type used. Access to a greater number of cell culture conditions on a
high-throughput format enables the generation of a larger and more-predictive
portfolio of data for new chemical entities. Thus, we have optimized the architecture
of both the DataChip and the MetaChip platform for metabolism-induced
toxicity measurements to enable the screening of compounds against multiple cell
types cultured in 3-D matrices under different conditions on a single chip platform.
In the new embodiment of the DataChip, cells are immobilized within a variety of
matrices on 532 elevated pillars that are then submersed in microwells of the modified
complementary MetaChip. Recombinant enzymes are sequestered within matrices
in the microwells to enable the in-situ generation of metabolites. Because
each cell culture is immersed in media in discrete wells, multiple cell types requiring
different media can be cultured on the same chip. The IC50 values of compounds
known to have cytochrome P450 generated toxic metabolites, such as Troglitazone,
are compared under different culture conditions for several cell types. These studies
demonstrate the increased throughput and capability of the modified
MetaChip/DataChip system to identify metabolism-induced toxicity at early stages
in compound safety assessment.
2526 HIGH-THROUGHPUT GENE TRANSFECTION ON A
THREE-DIMENSIONAL (3-D) CELL MICROARRAY
PLATFORM FOR METABOLISM-INDUCED
TOXICOLOGY SCREENING.
S. Kwon 1 , J. Gray 2 , B. Ku 3 , J. Ryan 2 , D. Clark 4 , J. Dordick 1 and M. Lee 2 .
1 Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute,
Troy, NY, 2 Solidus Biosciences, Inc., San Francisco, CA, 3 Central R&D Institute,
Samsung Electro-Mechanics Co., Suwon, Republic of Korea and 4 Department of
Chemical Engineering, University of California Berkeley, Berkeley, CA.
Variation in metabolic enzyme expression among segments of the human population
may cause deviations from the expected pharmacokinetic profile of a drug, resulting
in idiosyncratic toxicity or a lack of efficacy. Cell lines that stably express individual
or combinations of metabolic enzymes are emerging tools for prediction of
these rare clinical events; however, it is difficult to create and maintain a library of
stable cell lines that mimics the diversity of metabolic profiles that give rise to unexpected
outcomes. In addition, it is difficult to control the expression levels of
each of the enzymes in stably-transfected cell lines. To address this need, we have
developed a technique for simultaneously infecting an array of miniaturized 3-D
cell cultures with varying concentrations of recombinant adenoviruses carrying
genes for different metabolic enzymes, which generates an array of cell cultures with
differentiated metabolizing capabilities. Compounds can be added to this array of
cell cultures to assay the compounds and their metabolites for a variety of end
points including, but not limited to, cell viability. We have demonstrated the controlled
expression of individual and multiple reporter proteins (GFP and RFP) and
drug-metabolizing enzymes (CYP3A4, CYP2C9, and UGT1A4) in two human
liver cell lines (HepG2 and THLE-2 cells) on the chip by altering the MOI of the
various recombinant adenoviruses. The microarrays of 3-D human cells expressing
metabolic enzymes were tested with model compounds, such as acetaminophen, to
simulate enzyme-specific hepatotoxicity. These studies demonstrate that the new
chip platform can provide critical information necessary for evaluating metabolisminduced
toxicity in a high-throughput manner.
2527 CELL TOXICITY AND INTERLEUKIN (IL) 8
PRODUCTION: UNIQUE RELATIONSHIP WITH
ALLERGENICITY.
I. Kimber1 , C. F. Portsmouth1 , R. Pendlington2 , G. Maxwell2 and R. J.
Dearman1 . 1University of Manchester, Manchester, United Kingdom and 2Unilever Safety & Environmental Assurance Centre, Bedford, United Kingdom.
There has been considerable interest in the development of in vitro tests for the
characterisation of chemical allergens, such as the production by dendritic cell
(DC)-like cell lines of cytokines. These methods are often limited by toxicity and
delivery of chemical allergen in culture. We have constructed toxicity and IL-8 secretion
dose response curves for a number of human cell lines (DC-like : THP-1,
U937; erthyromyeloid: K562). Cells were incubated for 24h with the contact allergen
dinitrochlorobenzene (DNCB) or its water soluble analogue, dinitrobenzene
sulfonic acid (DNBS), or with the irritant benzenesulfonic acid (BS). All cell lines
expressed IL-8 constitutively and displayed similar toxicity profiles for each of the
individual chemicals. DNCB and DNBS enhanced IL-8 production at subtoxic
doses (10μM and 300μM, respectively). In contrast, despite using doses that
caused similar toxicity profiles to those observed for the allergens, there was no increase
in IL-8 secretion recorded for BS (30 to 300mM). Cells (THP-1 and U937)
were cultured with two additional allergens (dinitrothiocyanobenzene [DNTB]
and dinitrofluorobenzene [DNFB]) or two irritants (1-bromobutane [1BB] and
sodium lauryl sulfate [SLS]) at subtoxic and toxic doses. In addition, cells were incubated
at varying osmotic pressures (ranging from 0 to 500mOsMo). Treatment
with subtoxic doses of DNTB and DNFB (10μM) induced the production of IL-8
by both THP-1 and U937 cells, whereas toxic doses of 1BB and SLS (10μM and
0.3μM, respectively) were without effect. Furthermore, under conditions of hypoosmolality
(100mOsMo) and hyper-osmolality (400mOsMo), both of which
caused cell death, there was no marked increase in IL-8 secretion. These data reveal
that the relationship between toxicity and IL-8 production is unique to allergens.
Defining the signalling pathways involved may allow the identification of novel
cellular biomarkers that, compared with IL-8, may offer increased sensitivity and
selectivity.
2528 SCREENING DRUG-INDUCED MITOCHONDRIAL
TOXICITY USING A HIGH-THROUGHPUT DUAL-
PARAMETER CELL BASED ASSAY.
J. Hynes2 , C. Carey2 , S. Kirwan2 , R. Swiss1 and Y. Will1 . 1Compound Safety
Prediction, Pfizer Global Research & Development, Groton, CT and 2Luxcel Biosciences, Cork, Ireland.
Recent years have seen a growing appreciation of the importance of the mitochondrion
as a site for off-target drug effects. This in turn has prompted the development
of a variety of in vitro plate-based mitochondrial toxicity assays, including
measuring oxygen consumption in isolated rat liver mitochondria and quantifying
the differential ATP depletion rates of aerobically poised versus conventionally cultured
HepG2 cells. Both assays identify similar compounds, but each assay also
identifies a unique set of compounds. To investigate if a cell-based metabolic assessment
could be used to address shed light on this phenomenon, a 384 well assay capable
of simultaneous monitoring of both glycolytic flux and oxygen consumption
was developed. Cell-based analysis addresses the risk of over prediction which exists
with an isolated organelle while also allowing confirmation of mitochondrial dysfunction
by observing modulation of both oxygen consumption and glycolytic flux.
HL60s were chosen over an adherent cell model as they exhibit similar sensitivity to
mitochondrial impairment while also removing the requirement for pre-plating,
thereby simplifying workflows. The approach was initially validated using a small
compound set and subsequently used to screen a library of 200 compounds across a
10 point dilution series. A small subset of compounds is flagged by all three assays
with isolated mitochondrial analysis flagging more than ATP depletion. The vast
majority of compounds flagged by either assay are detected by cell-based metabolic
analysis; however this approach appears to be more sensitive, confirming metabolic
perturbations for a larger number of compounds, albeit typically above 125uM.
These data suggest that over-prediction using isolated mitochondria is not a significant
problem, but also points to the fact that additional retrospective analysis is required
to allow such cell-based metabolic analyses to be correctly interpreted.
2529 USING MULTIPLE ASSAYS TO ASSESS DRUG-
INDUCED MITOCHONDRIAL TOXICITY IN EARLY
TOXICOLOGY TESTING.
R. Swiss, P. Rana and Y. Will. Compound Safety Prediction, Pfizer Global Research
& Development, Groton, CT.
Drug-induced mitochondrial dysfunction has been shown to contribute to organ
toxicity and late stage attrition. Therefore, testing for drug-induced mitochondrial
dysfunction pre-clinically is vitally important. We compared two separate assays to
SOT 2011 ANNUAL MEETING 541