Cytotoxicity of monocrotaline in isolated rat hepatocytes: effects of dithiothreitol and fructose.
ABSTRACT Monocrotaline (MCT) is a pyrrolizidine alkaloid present in plants of the Crotalaria species that causes cytotoxicity and genotoxicity, including hepatotoxicity in animals and humans. It is metabolized by cytochrome P-450 in the liver to the alkylating agent dehydromonocrotaline (DHM). In previous studies using isolated rat liver mitochondria, we observed that DHM, but not MCT, inhibited the activity of respiratory chain complex I and stimulated the mitochondrial permeability transition with the consequent release of cytochrome c. In this study, we evaluated the effects of MCT and DHM on isolated rat hepatocytes. DHM, but not MCT, caused inhibition of the NADH-linked mitochondrial respiration. When hepatocytes of rats pre-treated with dexamethasone were incubated with MCT (5 mM), they showed ALT leakage, impaired ATP production and decreased levels of intracellular reduced glutathione and protein thiols. In addition, MCT caused cellular death by apoptosis. The addition of fructose or dithiotreitol to the isolated rat hepatocyte suspension containing MCT prevented the ATP depletion and/or glutathione or thiol oxidation and decreased the ALT leakage and apoptosis. These results suggest that the toxic effect of MCT on hepatocytes may be caused by metabolite-induced mitochondrial energetic impairment, together with a decrease of cellular glutathione and protein thiols.
- SourceAvailable from: Flávia Thomaz Verechia Pereira[Show abstract] [Hide abstract]
ABSTRACT: Abamectin (ABA), which belongs to the family of avermectins, is used as a parasiticide; however, ABA poisoning can impair liver function. In a previous study using isolated rat liver mitochondria, we observed that ABA inhibited the activity of adenine nucleotide translocator and F(o)F(1)-ATPase. The aim of this study was to characterize the mechanism of ABA toxicity in isolated rat hepatocytes and to evaluate whether this effect is dependent on its metabolism. The toxicity of ABA was assessed by monitoring oxygen consumption and mitochondrial membrane potential, intracellular ATP concentration, cell viability, intracellular Ca(2+) homeostasis, release of cytochrome c, caspase 3 activity and necrotic cell death. ABA reduces cellular respiration in cells energized with glutamate and malate or succinate. The hepatocytes that were previously incubated with proadifen, a cytochrome P450 inhibitor, are more sensitive to the compound as observed by a rapid decrease in the mitochondrial membrane potential accompanied by reductions in ATP concentration and cell viability and a disruption of intracellular Ca(2+) homeostasis followed by necrosis. Our results indicate that ABA biotransformation reduces its toxicity, and its toxic action is related to the inhibition of mitochondrial activity, which leads to decreased synthesis of ATP followed by cell death.Toxicology in Vitro 11/2012; · 2.65 Impact Factor
Cytotoxicity of monocrotaline in isolated rat hepatocytes: Effects of
dithiothreitol and fructose
Marcos A. Maiolia, Larissa C. Alvesa, Diego Perandina, Andréa F. Garciaa, Flávia T.V. Pereirab,
Fábio E. Mingattoa,*
aLaboratório de Bioquímica Metabólica e Toxicológica, UNESP-Univ Estadual Paulista, Campus de Dracena, 17900-000 Dracena, SP, Brazil
bLaboratório de Morfofisiologia da Placenta e Embrião, UNESP–Univ Estadual Paulista, Campus de Dracena, 17900-000 Dracena, SP, Brazil
a r t i c l e i n f o
Received 16 December 2010
Received in revised form 7 April 2011
Accepted 12 April 2011
Available online 21 April 2011
a b s t r a c t
Monocrotaline (MCT) is a pyrrolizidine alkaloid present in plants of the Crotalaria species
that causes cytotoxicity and genotoxicity, including hepatotoxicity in animals and humans.
It is metabolized by cytochrome P-450 in the liver to the alkylating agent dehy-
dromonocrotaline (DHM). In previous studies using isolated rat liver mitochondria, we
observed that DHM, but not MCT, inhibited the activity of respiratory chain complex I and
stimulated the mitochondrial permeability transition with the consequent release of
cytochrome c. In this study, we evaluated the effects of MCT and DHM on isolated rat
hepatocytes. DHM, but not MCT, caused inhibition of the NADH-linked mitochondrial
respiration. When hepatocytes of rats pre-treated with dexamethasone were incubated
with MCT (5 mM), they showed ALT leakage, impaired ATP production and decreased
levels of intracellular reduced glutathione and protein thiols. In addition, MCT caused
cellular death by apoptosis. The addition of fructose or dithiotreitol to the isolated rat
hepatocyte suspension containing MCT prevented the ATP depletion and/or glutathione or
thiol oxidation and decreased the ALT leakage and apoptosis. These results suggest that the
toxic effect of MCT on hepatocytes may be caused by metabolite-induced mitochondrial
energetic impairment, together with a decrease of cellular glutathione and protein thiols.
? 2011 Elsevier Ltd. All rights reserved.
Monocrotaline (MCT), a pyrrolizidine alkaloid phyto-
toxin, has well-documented hepatic and cardiopulmonary
toxicity for animals, including ruminants and humans
(Mclean,1970; Mattocks,1986; Huxtable, 1989; Souza et al.,
1997; Schultze and Roth, 1998; Stegelmeier et al., 1999;
Nobre et al., 2004, 2005). This compound is frequently
intentionally in the form of herbal medicine preparations
(Huxtable, 1989). It has been reported that its toxicity
depends on cytochrome P-450 mediated bioactivation to the
reactive pyrrolic metabolite dehydromonocrotaline (DHM)
(Butler et al.,1970; Lafranconi and Huxtable,1984; Roth and
Reindel, 1990; Wilson et al., 1992; Pan et al., 1993; Schultze
and Roth, 1998). This metabolite, despite having a half-life
of only a few seconds in aqueous media, is a powerful
alkylating agent that binds to DNA and proteins (Petry et al.,
1984; Hincks et al., 1991; Niwa et al., 1991; Wagner et al.,
1993; Yan and Huxtable,1995; Lamé et al., 2005).
We previously demonstrated that DHM, but not MCT,
inhibits the activity of NADH-dehydrogenase when added
at micromolar concentrations to isolated rat liver mito-
chondria, an effect associated with significantly reduced
ATP synthesis (Mingatto et al., 2007). Because the activity
of complex I is regulated by thiol groups, it was suggested
* Corresponding author. Tel.: þ5518 38218200; fax: þ5518 38218208.
E-mail address: firstname.lastname@example.org (F.E. Mingatto).
Contents lists available at ScienceDirect
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Toxicon 57 (2011) 1057–1064
that the inhibition of complex I NADH oxidase activity
resulted from oxidation of cysteine thiol groups by DHM. In
a recent study, we also demonstrated that DHM induces
membrane permeability transition (MPT) and the release of
cytochrome c associated with oxidation of protein thiol
groups in isolated rat liver mitochondria (Santos et al.,
It is well known that the thiol group in proteins and
non-proteins is involved in the maintenance of various
cellular functions. Some investigators have indicated that
protein thiols, more than non-protein thiols, are essential
for the maintenance of cell viability during exposure to
toxic chemicals (Nicotera et al., 1985; Nakagawa and
Moldéus, 1992). The liver removes the xenobiotics from
the body by the triad of actions oxidation (phase I),
conjugation (phase II) and elimination (phase III), but in
a few cases either phase I or phase II reactions can result in
more toxic species (Boelsterli, 2007). The metabolism of
MCT seems to be one of these cases. Within this context, in
the present study, we evaluated the mechanisms respon-
sible for MCT toxicity in isolated rat hepatocytes and the
roles of its metabolism, thiol groups and mitochondria.
2. Material and methods
The MCT was purchased from Sigma-Aldrich (St. Louis,
MO), and DHM was prepared from MCT according to pub-
lished procedures (Mattocks et al., 1989). The purity of the
resulting pyrrole was confirmed using NMR. All other
reagents were of the highest commercially available grade.
Dexamethasone was purchased from DEG, Brazil. Sodium
pentobarbital was a gift from Cristália, Brazil. MCT was
solubilized in 2 M HCl and was neutralized with 0.5 M
phosphate buffer. All stock solutions were prepared with
glass-distilled deionized water. MCT and DHM were dis-
solved in anhydrous dimethyl sulfoxide (DMSO).
Male Wistar rats weighing approximately 200 g were
used in this study. Animals were maintained at a maximum
of 4 rats per cage under standard laboratory conditions.
Water and food were given ad libitum. In experiments with
dexamethasone induction, rats were dosed intraperitone-
ally (50 mg/kg body weight) daily for 3 consecutive days
and used 24 h after the last dose. The experimental
protocols were approved by the Ethical Committee for the
Use of Laboratory Animals of the Universidade Estadual
Paulista “Júlio de Mesquita Filho”, Campus de Dracena.
2.3. Isolation and incubation of hepatocytes
For the surgical procedure, rats were anesthetized by
intraperitoneal injection of sodium pentobarbital (50 mg/
kg body weight). The hepatocytes were isolated by
collagenase perfusion of the liver as described previously
isolation was determined by Trypan blue (0.16%) uptake,
and initial cell viability in all experiments was more than
85%. Hepatocytes were suspended in Krebs-Henseleit
buffer, pH 7.4, containing 12.5 mM Hepes and 0.1%
albumin (BSA) and maintained at 4?C. Cells (1 ? 106/mL)
were incubated in 25-mL Erlenmeyer flasks kept under
constant agitation (30 rpm) at 37?C under a 95% O2and
5% CO2 atmosphere. Reactions were initiated by the
addition of MCT. Aliquots (0.5 mL) of the suspension were
removed from the mixture at appropriate times for the
determination of cell death and biochemical parameters.
In some experiments, cells were incubated with 20 mM
fructose or 10 mM dithiotreitol (DTT) 15 min before the
addition of MCT.
2.4. Oxygen uptake
Oxygen uptake by the isolated hepatocytes was moni-
tored polarographically with an oxygraph equipped with
a Clark-type oxygen electrode (Strathkelvin Instruments
Limited, Glasgow, Scotland, UK) at 37?C. Respiration buffer
contained 250 mM sucrose, 2 mM KH2PO4, 10 mM HEPES,
pH 7.2, 0.5 mM EGTA, 0.5% bovine serum albumin, and
5 mM MgCl2. Cells were treated with 0.002% digitonin, and
state 4 and state 3 mitochondrial respiration rates were
measured in the presence of 1 mg/mL oligomycin and 2 mM
ADP, respectively (Moreadith and Fisckum,1984). MCT and
DHM were added in the medium, immediately after the
initiation of state 3 respirations by ADP.
2.5. Evaluation of cell viability
Cell viability was assessed by the leakage of alanine
transaminase (ALT) from hepatocytes. Cell suspensions
were centrifuged (50 ? g for 5 min). ALT in the supernatant
was determined using an Alanine Transaminase Activity
Assay Kit (Bioclin, Quibasa, Brazil) according to the manu-
facturer’s instructions. Absorbance was measured at
340 nm with a DU-800 spectrophotometer (Beckman
Coulter Inc., Fullerton, CA, USA). Enzyme activity in the
supernatant is expressed as a percentage of the total
activity, which was determined bylysing the cells with 0.5%
2.6. Cell ATP content
Cell ATP was determined by means of the firefly
luciferin-luciferase assay system. The cell suspension was
centrifuged at 50 ? g for 5 min at 4?C, and the pellet
containing the hepatocytes was treated with 1 mL of ice-
cold 1 M HClO4. After centrifugation at 2000 ? g for
10 min at 4?C, aliquots (100 mL) of the supernatant were
neutralized with 70 mL of 2 M KOH, suspended in 100 mM
Tris-HCl, pH 7.8 (1 mL final volume), and centrifuged again.
Bioluminescence was measured in the supernatant with
a Sigma-Aldrich assay kit according to the manufacturer’s
instructions using a SIRIUS Luminometer (Berthold, Pforz-
2.7. Reduced glutathione (GSH) levels
The levels of GSH were determined by a fluorimetric
reaction with o-phthalaldialdehyde (OPT) (Hissin and Hilf,
M.A. Maioli et al. / Toxicon 57 (2011) 1057–1064
1976). The cell suspension was treated with 0.2 mL of 30%
TCA and centrifuged at 2000 ? g for 6 min. Aliquots
(100 mL) of the supernatant were mixed with 2 mL of
100 mM NaH2PO4buffer, pH 8.0, containing 5 mM EGTA.
One hundred microliters OPT (1 mg/mL) was added, and
fluorescence was measured 15 min later using the 350/
420 nm excitation/emission wavelength pair with RF-5301
PC fluorescence spectrophotometer (Shimadzu, Tokyo,
Japan). The values are expressed as nanomoles of GSH/106
cells using a standard curve. A blank with DTT was per-
formed to eliminate its interference in the fluorescence
2.8. Protein thiols
Protein thiol groups were determined using Ellman’s
reagent according to Sedlak and Lindsay (1968) with some
modifications. A sample (0.5 mL) of cell suspension was
centrifuged at 50 ? g for 5 min and the supernatant was
discarded. The cell pellet was treated with 1 mL of 5% tri-
chloroacetic acid, 5 mM EDTA. The protein precipitate was
washed twice with the same trichloroacetic acid-EDTA
solution. When DTT was used, this procedure was
repeated four times. Protein was redissolved in 3 mL of
0.1 M Tris-HC1 buffer, pH 7.4, containing 5 mM EDTA and
0.5% sodium dodecyl sulfate. Aliquots of this solution were
reacted with 0.1 mM (final concentration) 5,50-dithiobis(2-
nitrobenzoic)acid (DTNB) in 2 mL of Tris-EDTA buffer, pH
8.6. Absorption was measured at 412 nm and subtracted
from blank value obtained by treating sample aliquots with
5 mM N-ethylmaleimide before reaction with DTNB. The
values are expressed as nanomoles of –SH equivalents/106
cells using GSH as a standard.
2.9. Apoptosis assays
Cell death by apoptosis was determined by observing
morphological changes in the nuclei of cells incubated with
the fluorescent dye Hoechst 33342 (Kurose et al., 1997).
Samples (200 mL) were collected and centrifuged at 50 ? g
for 5 min, and the supernatants were discarded; the pellet
was suspended in Krebs/Henseleit medium, pH 7.4, and
incubated with 8 mg/mL of Hoechst 33342 for 15 min at
room temperature. After incubation, the samples were
centrifuged twice at 50 ? g for 5 min to remove excess dye.
After the washes, the cells were suspended in 100 mL of
Krebs/Henseleit medium, pH 7.4. Cells were analyzed with
a fluorescence microscope (DM 2500 type, Leica, Rueil-
Malmaison, France), and the percentage of apoptotic cells
was quantitated using QWin software.
2.10. Statistical analysis
Comparisons of the several treated groups and the
relevant controls were made by analysis of variance
(ANOVA) followed by Dunnett’s test. Comparisons between
multiple groups were made using Newman–Keuls’s test
implemented in GraphPad Prism software, version 4.0 for
Windows (GraphPad Software, San Diego, CA, USA). Values
of P < 0.05 were considered significant.
3.1. Effects of MCT and DHM on the respiration of
mitochondria in isolated rat hepatocytes
Fig. 1 shows the inhibitory effect of DHM on glutamate
plus malate-supported state 3 (ADP-stimulated)respiration
of mitochondria in digitonin-permeabilized hepatocytes.
The effect was immediate and concentration-dependent,
beginning at 50 mM DHM; the parent compound MCT did
not inhibit state 3 respiration even at a concentration of
2 mM (Fig. 1). Neither MCT nor DHM stimulated state 4
(basal) respiration (results not shown). These results indi-
catethatthe metaboliteDHM inhibits the respiratorychain,
whereas neither the parent compound nor the metabolite
effectively uncouples the oxidative phosphorylation of
mitochondria, as assessed in isolated hepatocytes. These
results are in agreement with those previously described
in isolated mitochondria (Mingatto et al., 2007).
3.2. Toxic effects of MCT on isolated rat hepatocytes
The incubation of MCTat concentrations such as 10 mM
with hepatocytes isolatedfromnormal rats did not produce
toxic effects (results not shown). Thus, in order to stimulate
the production of MCT metabolites by isolated hepatocytes,
rats were previously treated with dexamethasone, an
inducer of cytochrome P-450 3A (Gonzales, 1990). Metab-
olism of MCT has been attributed to this cytochrome (Reid
et al., 1998). The addition of increasing concentrations of
MCT to hepatocytes of rats pre-treated with dexametha-
sone resulted in decreased cell viability, as assessed by ALT
leakage into the incubation medium (Fig. 2A). ALT leakage
was concentration- and time-dependent, with a significant
increase being observed at MCT concentrations of 5 and
dromonocrotaline (DHM) on glutamate plus malate-supported state 3 (ADP-
stimulated) respiration of mitochondria in digitonin-permeabilized isolated
rat hepatocytes. The figure is representative of four experiments with
different cell preparations. *,**Significantly different from control (absence
of MCT or DHM) (P < 0.05 and P < 0.01, respectively).
1. Effects ofmonocrotaline(MCT)or itsmetabolitedehy-
M.A. Maioli et al. / Toxicon 57 (2011) 1057–1064
7.5 mM at 60 min incubation. In a previous report, we
showed that the exposure of isolated perfused liver to MCT
results in bioenergetic metabolism failure, which may
reflect cell death due to decreased cellular ATP (Mingatto
et al., 2008). In the current study, the incubation of iso-
lated hepatocytes with MCT promoted a gradual decrease
in ATP levels, which appeared to correlate closely with cell
death (Fig. 2B). After 90 min incubationwith 5 mM or more
of drug, when almost all cells lost viability, ATP was almost
3.3. Effects of fructose and DTT on MCT-induced injury of
isolated rat hepatocytes and ATP levels
In order to investigate the mechanisms involved in the
cytotoxicity of MCT we evaluated the protective effects of
fructose (20 mM) and DTT (10 mM) using 5 mM MCT.
Fructose is an efficient substrate for glycolytic ATP forma-
tion in hepatocytes and protects against the loss of cell
viability due to mitochondrial impairment. Such protection
implies that cytotoxicity involves the inhibition of non-
glycolytic mitochondrial ATP formation (Mingatto et al.,
2002). Pre-treatment of hepatocytes with fructose pre-
vented the decrease of cell viability caused by MCT
(Fig. 3A). After 15 min pre-incubation with fructose, the
intracellular levels of ATP were decreased to 30% of the
control levels. This effect would be a consequence of the
action of fructokinase producing fructose-1-phosphate
with ATP consumption (Nakagawa et al., 1996). The addi-
tion of MCT did not further decrease the ATP levels, which
remained constant for the 90-min incubation period
(Fig. 3B). Because protein thiols and cellular thiol groups
have long been described as important targets for reactive
intermediates derived from some chemicals including MCT
(Moore et al., 1985; Reed, 1990; Yan and Huxtable, 1996;
Lamé et al., 2005), we also investigated the effects of DTT,
a thiol reductant, on the MCT-induced cytotoxicity. Both
the cytotoxicity and loss of intracellular ATP caused by
5 mM MCT were prevented by the addition of DTT (Fig. 3A
and B, respectively).
3.4. Effects of MCT on intracellular levels of glutathione and
protein thiols and protection by fructose and DTT
The oxidative status of hepatocytes in the presence of
MCT (5 mM) was evaluated by measuring the levels of GSH
and protein thiol. We observed a time-related decrease in
these parameters (Figs. 4 and 5, respectively), with the GSH
level being depleted more rapidly than that of protein
MCT 1 mM
MCT 2.5 mM
MCT 5 mM
MCT 7.5 mM
0 30 60 90
Fig. 2. Time course of the decrease in cell viability (A) and ATP levels (B) in
isolated rat hepatocytes incubated without (Control) or with different
concentrations of monocrotaline (MCT). Results are shown as the mean
?S.E.M. of four experiments with different cell preparations. *,**Significantly
different from “without MCT” for the corresponding time points (P < 0.05
and P < 0.01, respectively).
C ont rol
MC T + F
MC T + DT T
ALT l eakage
(% o f t ot al )
0 30 60 90
Ti me ( mi n)
(n mo l/ 10 6 c e lls )
Fig. 3. Effects of 20 mM fructose (F) and 10 mM dithiotreitol (DTT) on the
5 mM monocrotaline (MCT)-induced decrease in cell viability (A) and ATP
levels (B) in isolated rat hepatocytes. Results are shown as the mean ?S.E.M.
of four experiments with different cell preparations. *,**Significant differ-
ences between the group of MCT alone and the group of MCT plus F or MCT
plus DTT (P < 0.05; P < 0.01, respectively).
M.A. Maioli et al. / Toxicon 57 (2011) 1057–1064
thiols. As shown in Fig. 4, DTTcaused a significant decrease
in GSH oxidation induced by MCT, and fructose was unable
to prevent this effect. Pre-incubation with DTT significantly
inhibited the oxidation of protein thiol groups caused by
MCT; however, in the cells that were previously incubated
with fructose, we did not observe any protection (Fig. 5).
3.5. Protective effect of fructose and DTT on MCT-induced
Fig. 6 shows that MCT induces programmed cell death.
After 60 min of incubation, the cell suspension that
received only MCT showed a significant increase in the
number of apoptotic cells compared to the control cells
(without the addition of MCT). When the hepatocytes were
incubated with 20 mM fructose or 10 mM DTT prior to MCT
(5 mM) treatment, however, a lower frequency of apoptotic
cells was observed, and this protection was evident until
the end of the incubation period (90 min).
MCT, a pyrrolizidine alkaloid phytotoxin, has well-
documented hepatotoxicity both for animals and humans
et al.,1999; Nobre et al., 2004, 2005). Cytochrome P-450 in
the liver bio-activates MCT to an alkylating pyrrole deriv-
ative, DHM, which is considered responsible for the toxic
effects of MCT (Butler et al.,1970; Lafranconi and Huxtable,
1984; Roth and Reindel, 1990; Pan et al., 1993). Previously,
we have demonstrated that DHM, but not MCT, is toxic to
respiration dysfunction (Mingatto et al., 2007). Further-
more, we have also shown that the exposure of isolated
perfused liver of phenobarbital-treated rats to MCT results
in bioenergetic metabolism failure, which may reflect cell
death due to decreased cellular ATP (Mingatto et al., 2008).
In addition, we demonstrated that DHM can promote
cellular apoptosis by inducing MPT and cytochrome c
release (Santos et al., 2009).
MCT + F
MCT + DTT
1 / l o
) s l l e
Fig. 4. Effects of 20 mM fructose (F) and 10 mM dithiotreitol (DTT) on the 5 mM monocrotaline (MCT)-induced decrease in GSH level in isolated rat hepatocytes.
Results are shown as the mean ?S.E.M. of four experiments with different cell preparations. **Significantly different from Control (C) obtained without MCT
(P < 0.01).###Significantly different from MCT (P < 0.001).
MCT + F
MCT + DTT
- n i e t o
1 / l o
) s l l e
Fig. 5. Effects of 20 mM fructose (F) and dithiotreitol (DTT) on the 5 mM monocrotaline (MCT)-induced decrease in protein thiols level in isolated rat hepatocytes.
Results are shown as the mean ?S.E.M. of four experiments with different cell preparations. *Significantly different from Control (C) without MCT (P < 0.05).
#Significantly different from MCT (P < 0.05).
M.A. Maioli et al. / Toxicon 57 (2011) 1057–1064
GSH is present in most cells, and it is the most abundant
thiol in the intracellular medium (Meister and Anderson,
1983). Its activity in the cell may be to scavenge chemical
compounds and their metabolites by enzymatic and
substances before they can react at nucleophilic sites crit-
ical to cell viability (De Bethizy and Hayes, 2001). It may
also act as a substrate for glutathione peroxidase, thereby
reducing the destruction caused by free radicals and
xenobiotics (Reed, 1990). After treatment of hepatocytes
with MCT it was observed that the GSH levels were
drastically reduced, and by adding DTT, a thiol reducing
compound (Nicotera et al., 1985) at a concentration of
10 mM, no change was observed in GSH levels, protecting
the cells. Accordingly, sulfur-containing amino acids such
as cysteine and taurine have been shown to suppress the
toxic effects of MCT (Hayashi and Lalich, 1968; Yan and
A link between reduced protein thiol levels and cyto-
toxicity has been demonstrated in a study conducted with
the chemical menadione (Di Monte et al., 1984). In our
laboratory, studies with isolated mitochondria showed that
Fig. 6. (Top panel) Representative figures showing effects of 20 mM fructose (F) and 10 mM dithiotreitol (DTT) on the 5 mM monocrotaline (MCT)-induced
apoptosis in isolated rat hepatocytes (Hoescht staining) after 90 min of incubation from the four experimental groups i.e. Control (A), MCT (B), MCTþDTT (C) and
MCTþF (D) (N ¼ normal cells; Apop. ¼ apoptotic cells). (Bottom panel) Quantitation of apoptotic cells expressed as the percentage of total cells counted. Results
are shown as the mean ?S.E.M. of four experiments with different cell preparations. **Significantly different from Control (C) without MCT (P < 0.01).
##Significantly different from MCT (P < 0.01).
M.A. Maioli et al. / Toxicon 57 (2011) 1057–1064
DHM, but not MCT, has the ability to oxidize protein thiol
groups (Santos et al., 2009). Therefore, to investigate
whether this would also happen in hepatocytes, we incu-
bated the isolated hepatocytes with MCT and observed
a significant oxidation of –SH groups of proteins at 90 min
of incubation. However, when DTT was added, the oxida-
tion of these groups was prevented.
Thiol groups, in addition to participating in the anti-
oxidant defense system previously mentioned, regulate
various aspects of cellular function. Among these is the
induction of cell death by apoptosis, an activity regulated
by the redox state of the thiol groups (Sato et al., 1995).
One of the pathways that mediate apoptosis is the mito-
chondrial pathway (Green and Reed, 1998; Lemasters
dependent inner mitochondrial membrane permeabiliza-
tion. This permeability of the inner membrane is associ-
ated with the opening of a pore called the permeability
transition pore. The opening of the pore results in the
potential loss of the mitochondrial membrane, swelling of
the mitochondria and rupture of the mitochondrial outer
membrane (Zoratti and Szabò, 1995; Halestrap et al.,
2002), and it is sufficient to promote the release of cyto-
chrome c (a component of the electron transport chain
that allows the transfer of electrons between complex III
and IV) into the cytoplasm of the cell (Kroemer, 1997).
Cytochrome c in turn interacts with apoptotic protease
activating factors (Apaf), triggering the cascade of activa-
tion of pro-caspases by proteolytic cleavage and causing
death by apoptosis.
By assessing the effects of MCT on the induction of
apoptosis with the dye Hoechst 33342 in parallel with
monitoring the decrease in cell viability by changes in the
pattern of release of the enzyme ALT, we found that MCT is
able to induce programmed cell death. A possible cause for
this observed effect can be found in our previous work
with isolated mitochondria (Mingatto et al., 2007). We
demonstrated that DHM inhibits NADH-dehydrogenase,
causing a significant reduction in the synthesis of ATP,
which is a critical event for the development of cell
damage by necrosis or apoptosis (Nicotera et al., 1998). In
addition, DHM causes the oxidation of thiol groups of
proteins from mitochondria, resulting in the release of
cytochrome c (Santos et al., 2009), which initiates the
cascade of induction of programmed cell death. Accord-
ingly, Copple et al. (2004) showed that MCT kills cultured
hepatic parenchymal cells by apoptosis, with activation of
Fructose is an efficient substrate for glycolytic ATP
formation and protects against cell death induced by toxic
compounds (Wu et al., 1990; Nieminen et al., 1994). It
protected the hepatocytes against significant programmed
cell death induced by MCT, demonstrating the important
role of reducing levels of ATP in this process. The protection
provided by DTT indicates that the oxidation of thiol groups
is also involved in the induction of apoptosis by MCT. Thus,
our results suggest that the metabolite-induced mito-
chondrial energetic impairment, together with a decrease
of cellular glutathione and protein thiol groups, can
contribute to the toxic effects of MCT on hepatocytes.
Conflict of interest statement
This work was supported by grants from Fundação de
Amparo à Pesquisa do Estado de São Paulo (FAPESP),
Process number 2004/09882-7, Brazil. The authors thank
Michele Costa Lima for technical assistance.
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