DCPT and its metabolites DCTA, MAA, and DPI were evaluated for their effects on cell viability and ATP content in HepG2 cells. DCTA was found to be the most toxic, causing over 50% decreases in both cell viability and ATP levels at a concentration of 200 uM. DCPT and DPI showed more modest toxicity, with decreases of 16.5-28% and 10-21.5% respectively. MAA only affected ATP levels, with a 16.9% decrease. The combination of MAA and DPI showed no toxic effects. These results suggest that metabolism of DCPT into DCTA may play a key role in the toxicity observed in vitro and in vivo.

1. Effect of 3-(3,5-Dichlorophenyl)-2-4-thiazolidinedione (DCPT) and its Metabolites on Cell Viability and
ATP Content in HepG2 Cells
Department of Pharmaceutical Sciences, University of the Sciences, Philadelphia, PA
Aubrey S. Perrine, Ruy Tchao, Peter J. Harvison
Abstract
Troglitazone was previously marketed for
treatment for diabetes, but caused
hepatotoxicity when it was widely used by
humans. A key structural component of this drug
is the thiazolidinedione (TZD) ring, which is
thought to contribute to its toxic nature.
Another TZD ring-containing compound, 3-(3,5-
dichlorophenyl)-2-4-thiazolidinedione (DCPT),
has been shown to be cytotoxic in HepG2 cells.
Metabolism of DCPT initially results in a
spontaneous hydrolysis reaction to produce
[[[3,5-dichlorophenyl)amino]carbonyl]thio]acetic
acid (DCTA), followed by further metabolism into
mercaptoacetic acid (MAA) and 3,5-
dichlorophenyl isocyanate (DPI). The overall
mechanism of DCPT toxicity is unknown, but it is
likely that mitochondrial damage is involved. The
purpose of this research is to further our insight
of the cytotoxic mechanism by evaluating the
early effects of DCPT and its metabolites on
cellular viability and ATP levels using HepG2
cells. Cellular viability depicts the degree of
cytotoxicity of the compound. ATP depletion
would suggest mitochondrial damage since most
cellular ATP is produced in the mitochondria.
Although decreased ATP levels may trigger
necrosis, it is difficult to distinguish necrosis
from apoptosis, especially since both could be
occurring simultaneously. To achieve our goals,
HepG2 cells were cultured in 96-well plates and
allowed to grow for 24 hours. The cells were
then treated with one of the four compounds, at
different concentrations (0, 25, 50, 100 or 200
uM) for 3 hours. Cytotoxicity was assessed using
commercially available assay kits. We found that
toxicity was both compound- and concentration-
dependent. For example, DCPT (200 uM) reduced
cell viability and ATP content by ca. 25%;
whereas DCTA (200 uM) produced profound
(>50%) decreases in both parameters. By
contrast, DPI and MAA had more modest effects
on cell viability and ATP content (<15% decreases
at 200 uM). In conclusion, both parameters were
useful for assessing potential cytotoxicity of
these compounds. Furthermore, DCPT and its
metabolites exhibited differential toxicity in
HepG2 cells. Further experiments will be needed
to determine if ATP depletion proceeds loss of
cell viability.
Supported by the USciences Department
of Pharmaceutical Sciences.
Background
Troglitazone
Troglitazone is a member
Of the thiazolidinedione (TZD)
class of drugs used for the
treatment of type II diabetes.
The distinguishing structural component of these drugs is
the TZD ring. Although TZDs are still used to treat
diabetic patients, troglitazone was shown to induce
hepatotoxicity and was taken off of the market shortly
after its release. The mechanism behind this toxicity is
still unknown, but it is thought that the TZD ring
structure plays a crucial role.
DCPT
Another TZD ring-containing compound is DCPT, which
has been shown to be cytotoxic in HepG2 cells, as well as
hepatotoxic in rats. DCPT undergoes a spontaneous
hydrolysis reaction on the TZD ring, giving rise to its
metabolite DCTA. A subsequent spontaneous or enzymatic
reaction cleaves DCTA into the downstream metabolites
MAA and DPI. DCPT and several of its metabolites have
been previously found to induce some degree of toxicity.
The mechanism behind this cytotoxicity is still under
investigation. To further understand this toxicity, we
evaluated DCPT, DCTA, MAA, and DPI for cellular viability
and ATP content using HepG2 cells. Since ATP levels are
tightly linked to the process of apoptosis, this parameter
is key to understanding the mode of cellular death.
Methodology
Cells
Human hepatoma cells (HepG2 WT) were cultured in
Dulbecco's modified eagle medium (DMEM/F12)
supplemented with penicillin/streptomycin and 10%
fetal bovine serum (FBS). Cells were incubated at 37°C
under 5% CO2.
Incubations
Cytotoxicity and ATP experiments were completed
with DCPT and its three metabolites: DCTA, MAA, and
DPI, as well as a combination incubation of MAA+DPI.
HepG2 WT cells were plated at 20,000 cells/well in
96-well plates (MTS: white strip-well, ATP: black-well)
in DMEM/F12 media and were allowed to grow for 24
hours at 37°C under 5% CO2. The cells were then
treated with each compound in Hanks’ balanced salt
solution (HBSS; containing up to 0.1% DMSO) at
concentrations of: 0, 25, 50, 100, and 200 µM.
Controls were incubated with HBSS (0.1% DMSO) only.
Cell Viability Assay
After the 3 hour treatment period, cytotoxicity was
evaluated using the MTS assay according to the
manufacturer’s protocol. Absorbance was measured at
492 nm using a Victor 3 model 1420 multilabel counter
(PerkinElmer, Inc., Wellesley, MA).
ATP Assay
Following the 3 hour treatment period, ATP levels
were evaluated using the CellTiter-Glo® Luminescent
Cell Viability Assay (Promega, Madison, WI) according
to the manufacturer’s protocol.
Statistical Analysis
Statistical analyses were conducted using SigmaPlot
13.0 software. Results are expressed as means ±
standard errors (n = 3-4). The data were analyzed by a
one way ANOVA, followed by a Student-Newman-Keuls
or Dunnett’s test. Differences in the means were
considered significant when p < 0.05.
Summary and Conclusions
• DCTA appears to induce the highest degree of toxicity
at a concentration as low as 100 uM, but more
prominently at 200 uM
• 50% loss in viability
• 92.5% loss in ATP
• DPI and DCPT showed a modest degree of toxicity
• 16.5% - 28% loss in viability
• 10% - 21.5% loss in ATP
• MAA affected ATP levels only: 16.9% loss
• The MAA-DPI combo showed no toxic effects
• Although DCPT and its metabolites showed some
degree of toxicity, it is DCTA that seems to be most
harmful
• Metabolism of DCPT into DCTA may be key in
producing the toxicity seen in vitro and in vivo.
Future Plans
• Time course experiments to measure the loss in ATP
levels over time, compared to cell viability loss
• ATP standard curve to quantitatively measure the
concentration of ATP that is being depleted
Results
References
• Kennedy EL, Tchao R, Harvison PJ (2011). Nephrotoxic and
hepatotoxic potential of imidazolidinedione-, oxazolidine-
dione, and thiazolidinedione-containing analogues of N-
(3,5-dichlorophenyl)succinimide in Fischer 344 rats.
Toxicology, 186: 79-91.
• Frederick D, Jacinto E, Patel N, Rushmore T, Tchao R,
Harvison P (2011). Cytotoxicity of 3-(3,5-dichlorophenyl)-
2,4-thiazolidinedione (DCPT) and analogues in wild type
and CYP3A4 stably transfected HepG2 cells. Toxicol. in
Vitro, 25: 2113–2119
* P < 0.05 versus control concentration
Metabolic Scheme