Toxicon 55 (2010) 13311337

Contents lists available at ScienceDirect

Toxicon
journal homepage: www.elsevier.com/locate/toxicon

Comparative effects of lantadene A and its reduced metabolite

on mitochondrial bioenergetics
Andrea F. Garcia a, Hyllana C.D. Medeiros a, Marcos A. Maioli a, Michele C. Lima a,
Bruno A. Rocha b, Fernando B. da Costa b, Carlos Curti c, Milton Groppo d, Fabio E. Mingatto a, *
a
rio de Bioqumica, Faculdade de Zootecnia, UNESPUniv Estadual Paulista, Campus Experimental de Dracena, Dracena, SP 17900-000, Brazil
Laborato
b
Departamento de Ciencias Farmaceuticas, Faculdade de Ciencias Farmaceuticas de Ribeira
o Preto, Universidade de Sa
o Paulo, Ribeira
o Preto, SP 14040-903, Brazil
c
Departamento de Fsica e Qumica, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Ribeira
o Preto, SP 14040-903, Brazil
d
Departamento de Biologia, Faculdade de Filosoa, Ciencias e Letras de Ribeirao Preto, Universidade de Sa o Paulo, Ribeira
o Preto, SP 14040-901, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Lantana (Lantana camara Linn.) is a noxious weed to which certain medicinal properties
Received 1 September 2009 have been attributed, but its ingestion has been reported to be highly toxic to animals and
Received in revised form 23 January 2010 humans, especially in the liver. The main hepatotoxin in lantana leaves is believed to be the
Accepted 3 February 2010
pentacyclic triterpenoid lantadene A (LA), but the precise mechanism by which it induces
Available online 10 February 2010
hepatotoxicity has not yet been established. This work addressed the action of LA and its
reduced derivative (RLA) on mitochondrial bioenergetics. At the concentration range
Keywords:
tested (525 mM), RLA stimulated state-4 respiration, inhibited state-3 respiration, cir-
Lantana camara
Lantadene A cumvented oligomycin-inhibited state-3 respiration, dissipated membrane potential and
Reduced lantadene A depleted ATP in a concentration-dependent manner. However, LA did not stimulate state-4
Hepatotoxicity respiration, nor did it affect the other mitochondrial parameters to the extent of its
Mitochondria reduced derivative. The lantadenes didnt inhibit the CCCP-uncoupled respiration but
Bioenergetics increased the ATPase activity of intact coupled mitochondria. The ATPase activity of intact
uncoupled or disrupted mitochondria was not affected by the compounds. We propose,
therefore, that RLA acts as a mitochondrial uncoupler of oxidative phosphorylation,
a property that arises from the biotransformation (reduction) of LA, and LA acts in other
mitochondrial membrane components rather than the ATP synthase affecting the mito-
chondrial bioenergetics. Such effects may account for the well-documented hepatoxicity of
lantana.
2010 Elsevier Ltd. All rights reserved.

1. Introduction Brito et al., 2004). However, despite its toxic effects,

Lantana (Lantana camara Linn.) is one of the most
poisonous weeds in the world. The noxious properties of
the plant are well documented: it causes cholestasis,
hepatotoxicity, photosensitization, and even fatality in
cattle, horses, sheep, dogs, and humans (Wolfson and
Solomons, 1964; Tokarnia et al., 1984; Black and Carter,
1985; Fourie et al., 1987; Sharma et al., 1988; Pass, 1991;
* Corresponding author. Tel.: 55 18 3821 8200; fax: 55 18 3821 8208.
E-mail address: fmingatto@dracena.unesp.br (F.E. Mingatto).
L. camara is extensively used in popular medicine because
of its anti-inammatory, antipyretic, antispasmodic, and
antibiotic properties (Sharma et al., 2007b).
The most well-known lantana compounds are the lan-
tadenes, which belong to the pentacyclic triterpenoid ole-
anane series. The most abundant triterpene acid is
lantadene A (LA); it has been implicated as the main culprit
responsible for the toxic effects of the plant (Sharma et al.,
1991, 2000, 2007b). Despite evidence that mitochondria
are affected by compounds present in lantana (Sharma
et al., 1982; Sharma, 1984), the mechanism by which it

0041-0101/$ see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2010.02.004
1332 A.F. Garcia et al. / Toxicon 55 (2010) 13311337

induces toxicity has not yet been clearly established. On the H3 C CH3
other hand, it was recently demonstrated that some lan-
tadenes exhibit in vitro and in vivo antitumor activity
O CH3
(Sharma et al., 2007a, 2008; Kaur et al., 2008). H
Mitochondria carry out a variety of biochemical O C C C
CH3
processes, but their main function is to produce a majority H3 C CH3 H
(>90%) of cellular ATP. The proton motive force, whose COOH
major impetus is the membrane potential (Dj) generated H
CH3
by electron transport along the respiratory chain in the
inner mitochondrial membrane, drives ATP synthesis via O
H
oxidative phosphorylation (Mitchell, 1961). Uncouplers of H3 C CH3
oxidative phosphorylation in mitochondria inhibit the LA
coupling between electron transport and phosphorylation
reactions and thus inhibit ATP synthesis (Terada, 1990).
They increase the permeability of the inner mitochondrial
membrane to protons along a gradient running from
H3 C CH3
intermembrane to matrix spaces; under this condition, the
organelle is no longer capable of sustaining ATP synthesis
(Kadenbach, 2003). It is believed that mitochondrial O CH3
H
uncoupling is a relevant mechanism for xenobiotic-induced O C C C
toxicity, particularly in the liver, which is the major site of H3C CH3 H CH3
xenobiotic uptake and metabolism (Wu et al., 1990). In COOH
addition, results from our research group suggest that H
CH3
mitochondria are the target organelle of toxic compounds
isolated from plants (Mingatto et al., 2007; Santos et al., HO
H
2009). H3C CH3
To date, no antidote to lantana toxicity is available, and
treatment of its symptoms has met with limited success RLA
(Sharma et al., 2007b). Thus, an understanding of the
Fig. 1. Chemical structures of lantadenes used in this study.
mechanism of lantana toxicity at the cellular and molecular
levels would help in the development of antidotes and
more rational therapies for lantana poisoning. In the
charcoal, which yielded a golden yellow extract. The solvent
present work, we addressed the actions of LA isolated from
was removed under reduced pressure, and the residue (15 g)
L. camara and its reduced derivative, known as RLA (Fig. 1),
was suspended in a methanol-H2O mixture (1:7, v/v) and
on mitochondrial bioenergetics by assessing their effects
extracted with chloroform (CHCl3, 2  40 mL). The organic
on respiration, membrane potential, ATP levels and ATPase
layer was dried over anhydrous Na2SO4 and the nal dried
activity in rat liver mitochondria. The inuences of lanta-
residue (6 g) was chromatographically puried over a silica
dene biotransformation on mitochondrial function and
gel column (180 g, 60120 mesh Merck, 7736) using CHCl3
liver toxicity are considered.
and CHCl3-methanol (99.5:0.5, v/v) as the mobile phase.
The lantadene-rich fraction was further puried and iso-
2. Materials and methods
lated by thin layer chromatography (silica gel PF254, 1 mm
thickness, Merck 7730) using CHCl3-methanol (99.5:0.5,
2.1. Plant material
v/v) as the eluting solvent (Sharma and Dawra, 1991) to
afford the known lantadene A and reduced lantadene A. The
L. camara Linn. (Lantana, family Verbenaceae) leaf
compounds were identied by nuclear magnetic resonance
samples were collected from a rural area in Monte Castelo
(NMR) of 1H and comparison with data from the literature
(21180 S, 513401000 W), Sao Paulo, Brazil. A voucher spec-
(Sharma et al., 1987, 1990). The purity of the compounds was
imen was identied (number SPFR 10364) by Prof. Milton
estimated by thin layer chromatography using different
Groppo and deposited in the herbarium of the Departa-
solvent systems, as well as 1H NMR analysis.
mento de Biologia da Faculdade de Filosoa, Ciencias e
Letras de Ribeirao Preto, Universidade de Sao Paulo,
Ribeirao Preto, Sao Paulo, Brazil. 2.3. Animals

2.2. Extraction and isolation of lantadenes
The lantana leaf samples were dried in the shade at 37  C
and ground into powder in an electric grinder. To 400 g of
the powder, 2000 mL of methanol were added and the
material was macerated for 24 h at room temperature with
intermittent shaking. The extract was ltered through
a muslin cloth and decolorized with 70 g of activated
Male Wistar rats weighing approximately 200 g were
housed in plastic cages under regulated temperature
(20  C) and the light/dark cycle (12 h:12 h). They were fed
commercially pelleted rat food (Purina, Brazil). The exper-
imental protocols were approved by the Ethical Committee
for the Use of Laboratory Animals of the Faculdade de
Zootecnia, Universidade Estadual Paulista Julio de Mes-
quita Filho, Campus de Dracena.
 A.F. Garcia et al. / Toxicon 55 (2010) 13311337
2.4. Isolation of rat liver mitochondria
Mitochondria were isolated by standard differential
centrifugation (Pedersen et al., 1978). Rats were sacriced
by decapitation and the liver was removed immediately,
sliced into 50 mL of medium containing 250 mM sucrose,
1 mM EGTA, and 10 mM HEPES-KOH, pH 7.2, and homog-
enized three times for 15 s at 1-min intervals with a Potter
Elvehjem homogenizer. Homogenate was centrifuged at
770  g for 5 min and the resulting supernatant further
centrifuged at 9800  g for 10 min. Pellet was suspended in
10 mL of medium containing 250 mM sucrose, 0.3 mM
EGTA, and 10 mM HEPES-KOH, pH 7.2, and centrifuged at
4500  g for 15 min. The nal mitochondrial pellet was
suspended in 1 mL of medium containing 250 mM sucrose
and 10 mM HEPES-KOH, pH 7.2, and used within 3 h. The
mitochondrial protein concentration was determined by
a biuret assay with BSA as the standard (Cain and Skilleter,
1987).
2.5. Mitochondrial respiration assay
Mitochondrial respiration was monitored using a Clark-
type oxygen electrode (Strathkelvin Instruments Limited,
Glasgow, Scotland, UK). One milligram of mitochondrial
protein was added to 1 mL of respiration buffer containing
125 mM sucrose, 65 mM KCl, and 10 mM HEPES-KOH, pH
7.4, plus 0.5 mM EGTA and 10 mM K2HPO4, at 30  C. Oxygen
consumption was measured using 5 mM succinate
(2.5 mM rotenone) or 5 mM glutamate 5 mM malate as
respiratory substrates in the absence (state-4 respiration)
or the presence of 400 nmol ADP (state-3 respiration).
2.6. Estimation of mitochondrial membrane potential
The mitochondrial membrane potential (Dj) was esti-
mated spectrouorimetrically using 10 mM safranine O as
a probe (Zanotti and Azzone, 1980) and a model RF-5301 PC
Shimadzu uorescence spectrophotometer (Tokyo, Japan)
at the 495/586 nm excitation/emission wavelength pair.
Mitochondria (2 mg protein) energized with 5 mM succi-
nate (2.5 mM rotenone) were incubated in a medium
containing 125 mM sucrose, 65 mM KCl, 10 mM HEPES-
KOH, pH 7.4, and 0.5 mM EGTA (2 mL nal volume).
2.7. ATP quantication
The amount of ATP was determined using the rey
luciferinluciferase assay system (Lemasters and
Hackenbrock, 1976). After incubation in the presence of
lantadenes, the mitochondrial suspension (1 mg protein/
ml) was centrifuged at 9000  g for 5 min at 4  C, and the
pellet was treated with 1 mL of ice-cold 1 M HClO4. After
centrifugation at 14,000  g for 5 min at 4  C, 100 mL
aliquots of the supernatants were neutralized with 5 M
KOH, suspended in 100 mM TrisHCl, pH 7.8 (1 mL nal
volume), and centrifuged at 15,000  g for 15 min. The
supernatant was worked-up with a Sigma/Aldrich assay kit
according to the manufacturers instructions and measured
using a SIRIUS Luminometer (Berthold, Pforzheim,
Germany).
1333
2.8. Mitochondrial swelling in hyposmotic potassium
acetate medium
Mitochondria (0.45 mg protein) were incubated in
a medium containing 54 mM potassium acetate, 5 mM
HEPES-NaOH, pH 7.1, 0.1 mM EGTA, 0.2 mM EDTA, 0.1 mM
sodium azide, 0.1% bovine serum albumin, 15 mM atrac-
tyloside, 1 mM antimycin A, and 0.3 mM propranolol in
order to inhibit the inner membrane anion channel, fol-
lowed by 1 mM valinomycin and lantadenes, in a nal
volume of 1.5 mL (Mingatto et al., 2000). Swelling was
estimated from the decrease in absorbance at 540 nm using
a model DU-800 spectrophotometer (Beckman Coulter,
Fullerton, CA).
2.9. ATPase activity
The mitochondrial ATPase activity was measured in
intact (coupled and uncoupled) and in freeze-thawing
disrupted mitochondria according to the protocol of
Bracht et al. (2003) with modications. Intact mitochondria
(1 mg protein/ml) were incubated in a medium containing
125 mM sucrose, 65 mM KCl, and 10 mM HEPES-KOH, pH
7.4, plus 0.2 mM EGTA and 5 mM ATP for 20 min at 37  C, in
the absence (coupled) and presence (uncoupled) of 1 mM
carbonyl cyanide m-chlorophenyl hydrazone (CCCP), in
a nal volume of 0.5 mL. When disrupted mitochondria
were used as enzyme source, the medium contained
20 mM TrisHCl (pH 7.4). The reaction was started by the
addition of 5 mM ATP and stopped by the addition of ice-
cold 5% trichloroacetic acid. ATPase activity was evaluated
by measuring the released inorganic phosphate as
described by Fiske and Subbarow (1925) at 700 nm using
a model DU-800 spectrophotometer (Beckman Coulter,
Fullerton, CA).
2.10. Statistical analysis
Data are expressed as mean  s.e. mean, and statistical
differences were calculated by one-way analysis of variance
(ANOVA) followed by the Dunnetts test using GraphPad
Prism, version 4.0 for Windows, from GraphPad Software
(San Diego, CA, USA).
3. Results
3.1. Effects of lantadenes on mitochondrial respiration
Mitochondrial oxygen consumption was monitored in
the presence of different concentrations of the lantadenes.
The parameters assessed were: state-3 respiration
(consumption of oxygen in the presence of respiratory
substrate and ADP) and state-4 respiration (consumption of
oxygen after ADP has been exhausted). At the concentra-
tions tested (525 mM), RLA, but not LA, stimulated, in
a dose-dependent manner, state-4 respiration of mito-
chondria energized with either succinate, a respiratory
chain site II substrate (Fig. 2), or glutamate plus malate,
which are respiratory chain site I substrates (data not
shown). RLA also inhibited, in a dose-dependent manner,
state-3 respiration of succinate-energized mitochondria,
1334 A.F. Garcia et al. / Toxicon 55 (2010) 13311337

LA LA
30 75

*
20 50

10 25

(nmol O 2 . min-1 mg protein -1)

( nmol O 2 . min-1mg protein )
-1
State 4 r espir ation r ate

State 3 respiration rate

0 0

RLA RLA
30
75
**
20 **
** 50 **
**
*
10 **
25

0
0
0 5 10 15 20 25
0 5 10 15 20 25
Lantadene (M)
Lantadene (M)
Fig. 2. Effects of lantadenes on the state-4 respiration rate of succinate-
Fig. 3. Effects of lantadenes on the state-3 respiration rate of succinate-
energized rat liver mitochondria. The assay conditions are described in
energized rat liver mitochondria. The assay conditions are described in
Materials and methods. Values represent the mean  s.e. mean of three
Materials and methods. Values represent the mean  s.e. mean of three
experiments with different mitochondrial preparations. *,**Signicantly
experiments with different mitochondrial preparations. *, **Signicantly
different from control (*P < 0.05 and **P < 0.01).
different from control (*P < 0.05 and **P < 0.01).

whereas LA was only inhibitory at 25 mM (Fig. 3); a similar

pattern was observed for glutamate plus malate (data not
shown).
In order to investigate uncoupling of oxidative phos-
phorylation, state-4 respiration was induced by addition of
oligomycin (1 mg/mL), an inhibitor of ATP synthase (FoF1-
ATPase). Under such conditions, uncouplers are able to
increase oxygen consumption. RLA, but not LA, circum-
vented the oligomycin-imposed inhibition of mitochon-
drial state-3 respiration (Fig. 4A). Subsequent experiments
with CCCP-stimulated mitochondrial respiration were
performed to test the inhibitor effect of the compounds in
the respiratory chain or ATP synthase. Both compounds
didnt inhibit CCCP-uncoupled respiration indicating that
only oxidative phosphorylation was inhibited (Fig. 4B).
3.2. Effects of lantadenes on mitochondrial membrane
potential (Dj)
Fig. 5 shows the effects of lantadenes on Dj of
succinate-energized rat liver mitochondria. In accordance
with the results of mitochondrial respiration, LA only
signicantly dissipated Dj when present at 25 mM, whereas
RLA exhibited a signicant dose-dependent effect on this
parameter along the entire concentration range evaluated.
3.3. Effects of lantadenes on mitochondrial ATP levels
The effects of lantadenes on mitochondrial ATP levels
were evaluated under the conditions of the respiratory
assay 15 min after mitochondria were incubated with the
compounds (Fig. 6). In agreement with the results on
mitochondrial respiration and membrane potential, LA
only caused a signicant decrease in the mitochondrial ATP
levels at 25 mM whereas RLA again exhibited a signicant
dose-dependent effect on this parameter along the entire
concentration range evaluated.
3.4. Effects of lantadenes on mitochondrial swelling
in hyposmotic potassium acetate medium
In order to evidence the protonophoric properties of
RLA, we performed mitochondrial swelling in hyposmotic
potassium acetate medium. Fig. 7 shows that even at 25 mM
RLA didnt promote a mitochondrial swelling, indicating
that RLA is not working as the classical uncouplers like
CCCP.
3.5. Effects of lantadenes on ATPase activity
The effects of lantadenes on the ATPase activity were
measured in intact mitochondria either in the absence
(coupled) or in the presence of CCCP (uncoupled) and in
freeze-thawing disrupted mitochondria, as shown in Table
1. The ATPase activity of coupled mitochondria was
increased by both LA and RLA at 25 mM. The ATPase activity
of uncoupled or disrupted mitochondria was not signi-
cantly affected by lantadenes.
 A.F. Garcia et al. / Toxicon 55 (2010) 13311337
A ADP
Oligo
LA
RLA or CCCP
CCCP
B 75
KCN
50 MO 2
LA or RLA
1 min
Fig. 4. Effects of lantadenes (25 mM) on the oligomycin-inhibited state-3 (A)
and CCCP-uncoupled (1 mM) (B) respiration. Figure is representative of three
experiments with different mitochondrial preparations. The arrows indicate
the addition of the compounds. Oligo: oligomycin 1 mg/mL. KCN: potassium
cyanide 1 mM.
4. Discussion
The critical role played by mitochondria in the mainte-
nance of cellular energy metabolism has long been recog-
nized. Substrates dissolved in the mitochondria aqueous
matrix space are oxidized by corresponding dehydroge-
nases, which reduce NAD or FAD. Electron transport from
the oxidation of NADH and FADH2 to O2 is tightly coupled to
ATP synthesis. It occurs through protein-bound redox
centers, from complex I (NADH-coenzyme Q reductase) or II
(succinate-coenzyme Q reductase) to III (coenzyme cyto-
chrome e reductase) and then to IV (cytochrome e oxidase).
The free energy released by this transport is conserved by
pumping out protons resulting in an electrochemical H
gradient across the inner mitochondrial membrane. The
electrochemical potential of this gradient is then harnessed
for the synthesis of ATP by complex V (ATP synthase); this
process is known as oxidative phosphorylation. A wide
variety of compounds have been described as uncouplers of
oxidative phosphorylation (Wallace and Starkov, 2000).
They decrease the efciency of ATP production by
increasing the permeability of the inner membrane or
decreasing the degree of coupling of the proton motive force
and phosphorylation complexes. Such a malfunction in
mitochondrial bioenergetics instantaneously transforms
mitochondria from cellular powerhouses into a molecular
1335
LA
100
**
75
50
25
(% of control)
RLA
100
*
**
50
25
**
**
0
0 5 10 15 20 25
Lantadene (M)
Fig. 5. Effects of lantadenes on the membrane potential of succinate-
energized rat liver mitochondria. The assay conditions are described in
Materials and methods. Values represent the mean  s.e. mean of three
experiments with different mitochondrial preparations. *, **Signicantly
different from control (*P < 0.05 and **P < 0.01).
furnace, efciently wasting the metabolic energy of
substrates (Wallace and Starkov, 2000).
Mitochondrial dysfunction is a fundamental pathogenic
mechanism of several signicant toxicities in mammals,
especially those associated with the liver (Szewczyk and
Wojtczak, 2002; Amacher, 2005). Diminishing ATP levels
is critical to the development of necrosis (Nicotera et al.,
1998; Wallace and Starkov, 2000; Szewczyk and
Wojtczak, 2002). Thus, in order to assess the potential
involvement of mitochondria in the hepatotoxicity
observed in lantana poisoning, we compared the effects of
lantadene A (LA) and reduced lantadene A (RLA) on the
bioenergetics of rat liver mitochondria. LA, which is
believed to be responsible for the toxic effects of lantana
(Sharma et al., 1991, 2007b), exhibited a signicant inhibi-
tory effect on state-3 respiration, but only at the highest
concentration tested. On the other hand, RLA, which is
a biotransformation product of LA (Sharma et al., 2000),
caused a concentration-dependent stimulation of state-4
respiration and inhibition of state-3 respiration. In addi-
tion, it dissipated the mitochondrial membrane potential
and depressed ATP levels at all concentrations in the 5
25 mM range. The stimulation of state 4 by RLA occurred
even in the presence of oligomycin, an ATP synthase
inhibitor, showed that RLA acts as an uncoupler of oxidative
phosphorylation (Lude et al., 2008), however, the lack of
stimulating of mitochondrial swelling in hyposmotic
potassium acetate medium indicates that RLA is acting in
a different way as that expected to occur with a classical
1336 A.F. Garcia et al. / Toxicon 55 (2010) 13311337

LA Table 1
Effect of lantadenes on ATPase activity in intact mitochondria either in the
10.0
absence (coupled) or in the presence of CCCP (uncoupled) and in freeze-
thawing disrupted mitochondria.
7.5
Coupled Uncoupled Disrupted
mitochondria mitochondria mitochondria
5.0 * mmol. min1 mg protein1
Control 20.64  0.73 31.97  0.71 60.50  3.40
2.5 LA 25 mM 29.86  0.67* 35.41  3.99 59.64  3.55
RLA 25 mM 30.40  0.80* 35.84  1.17 61.58  1.78
( nmol . mg protein -1)

0.0 LA: lantadene A; RLA: reduced lantadene A.

*P < 0.05 relative to control.
ATP

they are acting as inhibitors of ATP synthase. To test this

hypothesis we performed experiments to evaluate the
RLA
effects of lantadenes in the ATPase activity using intact
10.0
(coupled and uncoupled) and freeze-thawing disrupted
mitochondria with excess of ATP, a condition in which the
7.5
enzyme operates in the reverse direction, hydrolyzing ATP
(Bracht et al., 2003). The stimulation of the ATPase activity
5.0 ** in intact coupled mitochondria and the lack of effect in the
**
** ** ATPase activity of uncoupled or disrupted mitochondria
2.5 indicate that RLA impairs energy metabolism probably due
its uncoupler property and suggest that the effect of LA
0.0 must be related to an effect in other mitochondrial
membrane components rather than the enzyme.
0 5 10 15 20 25
In line with our results pertaining to RLA, it has been
Lantadene (M)
reported that, when administered to Wistar rats, RLA cau-
Fig. 6. Effects of lantadenes on the ATP levels in succinate-energized rat ses liver intoxication characterized by jaundice, anorexia,
liver mitochondria. The assay conditions are described in Materials and constipation, polyuria, polydipsia, and accumulation of
methods. Values represent the mean  s.e. mean of three experiments predominantly conjugated bilirubin in the serum. The liver
with different mitochondrial preparations. *, **Signicantly different from
lesion is consistent with intrahepatic cholestasis and is
control (*P < 0.05 and **P < 0.01).
characterized by swelling of hepatocytes, individual cell
necrosis and biliary ductular hyperplasia (Pass et al., 1979).
uncoupler as CCCP, which depolarizes the mitochondrial
It has been also demonstrated in rats that RLA causes
inner membrane through a protonophoric effect (Nicholls,
hepatotoxicity through a toxic metabolite and that injury to
1982). A comparison of the structures of LA and RLA, in
bile canaliculi, which is an early lesion in the development
turn, suggests that 3-hydroxylation is a determining
of cholestasis, probably occurs due a decrease in the Mg2-
structural feature for the uncoupling activity exhibited by
ATPase activity that could be a consequence of ATP deple-
RLA.
tion, (Pass et al., 1981).
The inhibition of state-3 respiration by both RLA and LA
The present study shows that lantadenes, in general, are
using complex I and II substrates associated with their
potential perturbators of mitochondrial bioenergetics
inability to inhibit CCCP-uncoupled respiration indicates
through different mechanisms and potency, with RLA being
that they are not inhibitors of respiratory chain but that
more potent than LA. It shows, in particular, that the
reduced derivative of lantadene A depresses ATP levels via
uncoupling of oxidative phosphorylation, which, in turn,
dissipate the mitochondrial membrane potential. This
constitutes, therefore, a potential mechanism of the well-
documented lantana toxicity in liver cells.
C or RLA
Acknowledgements
A 0. 0 5

CCCP
1 min
Fig. 7. Effects of RLA (25 mM) on mitochondrial swelling in hyposmotic
potassium acetate medium. The assay conditions are described in Materials
and methods. Figure is representative of three experiments with different
mitochondrial preparations. The arrow indicates the addition of the
compounds. C: control, without addition of RLA or CCCP (1 mM).
This work was supported by grants from Fundaao de
Amparo a` Pesquisa do Estado de Sao Paulo (FAPESP),
PROPe-Unesp (Programa Primeiros Projetos) and Conselho
Nacional de Desenvolvimento Cientco e Tecnologico
(CNPq), Brazil. Results were presented by Andrea Fontes
Garcia to the Faculdade de Odontologia de Araatuba,
Universidade Estadual Paulista Julio de Mesquita Filho,
in partial fulllment of the requirements for the Master
degree in Ciencia Animal.
 A.F. Garcia et al. / Toxicon 55 (2010) 13311337
Conict of interest
The authors declare that there are no conicts of interest.
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