The Toxicologist - Society of Toxicology

iomarkers or observation of lesions at pathology in rodent studies. Cytochrome
P450s (CYPs) are measured as a surrogate of receptor activation, facilitating improved
interpretation of toxicity study findings. Therefore, liver samples were taken
from preclinical rat dose range finding (DRF) and 28 day toxicity studies and
analysed for levels of CYPs protein (CYP1A, CYP2B, CYP3A and CYP4A) by enzyme
linked immunosorbant assay (ELISA) using commercially available antibodies.
A review of 155 preclinical studies conducted between 2007 and 2009 and during
wich CYPs were analysed showed that a compound dependent increase in one
or more CYPs (> 3 fold above control) was observed in 26% of the studies. Among
these CYPs positive studies, 67% showed increased liver weight (> 25% of control
values), 32% showed increases in the liver function markers such as transaminases
ALT or AST (> 30% of control values), 35% showed decreases in exposure (> 30%
difference between last day and first day AUC) and 47% showed pathology findings
in the liver. The relatively low incidence rate of CYP induction observed in the
reviewed studies does not justify the inclusion of this endpoint as a routine analysis
in toxicity studies. However, CYP induction analysis should be used when liver dysfunction
is suspected from study findings as it can provide a valuable insign into the
mechanisms of toxicity. This might facilitate the clarification of differences in
species’ sensitivity during preclinical development and the determination of relevance
of these findings to man prior to clinical trials.
1910 A FLOW CYTOMETRIC MULTIPLEX HIGH-
THROUGHPUT APOPTOSIS ASSAY USING THE
HTFC SYSTEM.
C. Black 1 , B. Stout 1 , K. Luu 1 , T. Duensing 1 , P. Rana 2 and Y. Will 2 . 1 IntelliCyt
Corporation, Albuquerque, NM and 2 Compound Safety Prediction, Pfizer Inc.,
Groton, CT.
To determine the safety and efficacy of new drug targets, cell-based methodologies
are routinely utilized to screen large compound libraries. However, traditional cytotoxicity
assay methodologies often only provide single endpoints and single readouts.
Here we describe a novel high-throughput flow cytometry (HTFC) system
that can rapidly provide biological data at single cell resolution in both 96- and
384-well plate formats. Using this system, we have validated a multiplex apoptosis
screening assay that combines measurement of caspase 3/7 activation, cell viability,
and cell counts from each well. The assay had both a robust assay window (>80
fold) and Z’ values (> 0.7). Screening reproducibility was assessed using staurosporine
and resulted in intra- and inter-assay variation of less than 23%. A total
of 250 compounds were further investigated using the HTFC-based assay. These
compounds were tested from 300 μM to 0.3 μM and incubated against Jurkat cells
for 4 and 24 hours in a 384-well format. A ‘necrotic response signature’ was based
on large changes in cell counts within the well, a significant drop in viability, and
similarity to an apoptotic negative control such as Triton-X. We observed a variety
of apoptotic and necrotic inducing behaviors, for example, simvastatin at 300 μM
displayed a necrotic response signature similar to Triton-X based on a 10-fold drop
in cell count and a 50% loss in viability. Other similar necrotic compounds at 300
μM against Jurkat cells at the 24 hour time point were: progesterone, atomoxetine,
simvastatin, and tacrine. One clear example of an apoptotic compound through a
caspase 3/7 mechanism was amsacrine which resulted in a dose response without an
acute loss in cell count. The amsacrine EC50 values for caspase 3/7 activation were
12 μM at 4 hours and