The Toxicologist - Society of Toxicology

see whether both were needed or if one assay was sufficient in detecting mitochondrial
impairment. The first assay uses isolated rat liver mitochondria and measures
a compound’s effect on oxygen consumption. The second assay uses HepG2 cells
grown in either glucose or galactose containing media. Cells grown in galactose are
susceptible to mitochondrial insult, whereas cells grown in glucose are resistant. We
tested three sets of compounds that were associated with different organ-specific
toxicity. We tested 197 cardiac, 254 hepatic, and 83 nephritic compounds, which
included both, Pfizer internal and commercially available compounds.
Of the 534 compounds, 2.5 % flagged positive in isolated mitochondria as well as
in galactose grown HepG2 cells, 3.5% flagged in isolated mitochondria and glucose
and galactose cells, indicative of additional toxicity mechanisms. To our surprise
5% of the compounds (27 total) only showed toxicity in the isolated rat liver mitochondria
assay but not in HepG2 cells. We further analyzed these compounds using
the Seahorse platform which measures oxygen consumption rate and extracellular
acidification rate of intact cells simultaneously in real time. The mitochondrial impairment
was confirmed in the Seahorse experiments when compounds were analyzed
in real time. However, time course experiments revealed that cells could recover
from the insult and therefore exhibited no frank cell toxicity. In summary, the
current results indicate that including both assays in assessing mitochondrial toxicity
is important for identifying the greatest number of compounds showing mitochondrial
impairment. The mechanism behind the compensatory response warrants
additional future mechanistic work.
2530 JC-1 MITOCHONDRIAL MEMBRANE POTENTIAL
MEASUREMENTS OF H9C2 CELLS GROWN IN
GLUCOSE AND GALACTOSE CONTAINING MEDIA
DOES NOT PROVIDE ADDITIONAL PREDICTIVITY
TOWARDS MITOCHONDRIAL TOXICITY
ASSESSMENT.
P. Rana, Y. Will and S. Nadanaciva. Compound Safety Prediction, Pfizer Global
Research & Development, Groton, CT.
Drug-induced mitochondrial toxicity has been recognized to be a contributing factor
to a variety of organ toxicities. The fact that some, but not all members of a particular
drug class can induce mitochondrial dysfunction has necessitated the need
to deploy predictive screens within the drug development process. One of these
screens is a cell viability assay done in two types of media, one supplemented with
glucose and the other with galactose. Since galactose-grown cells are more susceptible
to mitochondrial toxicants than glucose-grown cells, this assay distinguishes
compounds that cause toxicity primarily through mitochondrial targets from those
that cause multifactorial toxicity. However, the assay cannot distinguish compounds
that cause toxicity through secondary effects on mitochondria from those
that cause toxicity through non-mitochondrial targets. Mitochondrial membrane
potential measurements are thought to be indicative of mitochondrial impairment
and can be measured in the absence of frank cytotoxicity. Here we investigated if
multiplexing mitochondrial membrane potential measurements with the fluorescent
dye, JC-1; together with cell viability measurements in glucose and galactosegrown
H9C2 cells could provide additional value for compounds that have a multifactorial
toxicity. We tested 29 drugs with well characterized mechanistic toxicity
profiles. These compounds set included compounds with different therapeutic uses
i.e. antidepressants, anti-diabetic agents, anti cancers, etc. The multiplexed assay
was able to detect mitochondrial toxicants correctly but did not reveal additional
information regarding compounds that exhibited multifactorial toxicity; hence, JC-
1 measurements did not provide additional information beyond what was detected
using the cell viability assay. Thus, we conclude that even though this multiplexed
assay is useful for HTS applications, it provides no additional value over the glucose-galactose
cell viability assay.
2531 TREATMENT OF FATTY ACIDS INCREASES THE
SUSCEPTIBILITY OF CELLS TO TOXIC COMPOUND
INDUCED INJURY.
Y. Luo, P. Rana and Y. Will. Compound Safety Prediction, Pfizer Global Research &
Development, Groton, CT.
Fatty acids are important source of energy and cells are exposed to various fatty
acids under physiological conditions. Excessive energy intake results in elevated levels
of fatty acids or triglycerides and cause metabolic disorders such as dyslipidemia,
obesity, insulin resistance and diabetes. Several lines of evidence suggest that metabolic
diseases increase the susceptibility to drug induced liver injury, cardiotoxicity
and renal toxicity. Regular cell culture conditions contain very low levels of fatty
acids, much less that than physiological concentrations. It has never been reported
whether fatty acids influence the sensitivity of cells to drug induced toxicity. The
aim of this study is to investigate the effects of fatty acids on drug induced toxicity.
542 SOT 2011 ANNUAL MEETING
In this study, we tested the effects of palmitate and oleic acid on the viability of
HepG2 cells. High concentrations of fatty acid treatment induced cell apoptosis
and reduced cell viability. Subsequntly, sub-toxic and toxic concentrations of fatty
acid were chosen to investigate whether fatty acids could potentiate drug induced
toxicity. We incubated the cells with various concentrations of fatty acids (palmitate
and oleic acid) and compounds followed by analyzing cell viability. Here we report
the results for statins (simvastatin, cerivastatin, lovastatin, atrovstatin, pravastatin)
and glitazones (ciglitazone, troglitazone, rosiglitazone, and pioglitazone).
Furthermore, increased population with metabolic diseases pose higher risk for
drug induced toxicity, and this assay may identify compounds that have higher risks
in this population, individually or in combination therapy.
2532 ER-STRESS AS A CONTRIBUTOR TO
HEPATOTOXICITY: DEVELOPMENT OF A SCREENING
PARADIGM.
T. Schroeter, L. Qui, M. Pletcher and Y. Will. Compound Safety Prediction, Pfizer
Global Research & Development, Groton, CT.
The greatest challenge facing the pharmaceutical industry today is developing more
effective decision making tools and strategies to reduce the attrition rate of novel
compounds during candidate selection and clinical development. Currently, approximately
24 out of 25 compounds fail in clinical trials, a situation that is unsustainable
for the industry. One of the major attrition reasons is hepatotoxicity. Gene
expression profiles associated with known idiosyncratic hepatotoxicity, revealed ER
stress as an important contributor to drug-induced liver toxicity. We developed two
assays to investigate whether induction of the endoplasmic reticulum stress response
(ERSR) is a predictive reporter of liver toxicity. The first assay detects nuclear
translocation of spliced XPB-1 by enzyme fragment complementation
(DiscoveRx) and the second assay is a luciferase CHOP reporter assay.
We screened 180 Pfizer internal and commercial available hepatotoxicants and 42
drugs not known to cause organ toxicity in both assays. Compounds were screened
in both assays at a final concentration of 150 uM. The XBP-1 screen identified 16
hits. Full dose response curves were generated for all hits and we were able to confirm
13 out of 16 XBP-1 hits. To our surprise, 4 (Ergocalciferol, Dipyridamole,
Orphenadrine and Propanolol) of the 13 confirmed XBP-1 hits were drugs with no
known organ toxicities. Of the 9 liver toxicants, 7 showed signs of cytotoxicity but
so did two of the 4 drugs not known to be organ toxic. The CHOP screen identified
3 hits which could all be validated. All of them are known liver toxicants
(Riluzole, Colchicine and Oxybendazole) with Riluzole showing signs of cytotoxicity.
Current work centers around confirmation of pathway activity of all active
compounds in HepG2 cells to assist in vitro to in vivo correlation.
2533 HIGH-CONTENT IMAGING ASSAY FOR DETECTING
ACCUMULATION OF COMPOUNDS IN LYSOSOMES.
S. Nadanaciva 1 , S. Lu 2 and Y. Will 1 . 1 Compound Safety Prediction, Pfizer Global
Research & Development, Groton, CT and 2 DSRD, Pfizer Global Research &
Development, La Jolla, CA.
Known as the cell’s recycling center, lysosomes are essential for the degradation of
old organelles, endotoxins and engulfed microbes. Many enzymes which function
in the acidic milieu of the lysosome are involved in degradative processes.
Compounds which are lipophilic weak bases or lipophilic neutral amines can readily
cross the lysosomal membrane from the cytoplasm. Once inside lysosomes,
however, these compounds become protonated and are unable to cross the lysosomal
membrane to re-enter the cytoplasm. The trapping of compounds within lysosomes
can cause alkalinization of these organelles, resulting in lysosomal dysfunction.
Hence, high-throughput screens to detect lysosomotropism, the accumulation
of compounds in lysosomes, are desirable.
In light of this, we developed a 96-well format multiplexed high content screening
(HCS) assay that measures both lysosomotropism and cytotoxicity. The effect of a
selection of compounds on these two endpoints was measured in H9c2 cells by
using the fluorescent dyes, LysoTracker® Red and Hoechst. Quantitative image
analysis showed that lysosomotropic compounds included the antimalarial, chloroquine,
antidepressants (sertraline, paroxetine, fluoxetine, imipramine, nortriptyline),
antipsychotics (chlorpromazine, thioridazine), antiarrhythmics (amiodarone,
propranolol), and some anticancer agents (tamoxifen, imatinib, dasatinib, gefitinib,
lapatinib). Although structurally and pharmacologically diverse, compounds
which caused lysosomotropism had a CLogP value > 2 and a basic pKa value between
6.5 to 11. In contrast, compounds which did not have this combination of
physicochemical properties were not lysosomotropic. The HCS assay that we developed
is a robust and rapid method for investigating lysosomotropism and can be
implemented within a screening paradigm for identifying drug-induced lysosomal
dysfunction.