Toxicon 55 (2010) 1331–1337

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
Andre´a 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, Fa´bio E. Mingatto a, *
a
´ rio de Bioquı´mica, Faculdade de Zootecnia, UNESP–Univ Estadual Paulista, Campus Experimental de Dracena, Dracena, SP 17900-000, Brazil
Laborato
b
Departamento de Cieˆncias Farmaceˆuticas, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira
˜o Preto, Universidade de Sa
˜o Paulo, Ribeira
˜o Preto, SP 14040-903, Brazil
c
Departamento de Fı´sica e Quı´mica, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Ribeira
˜o Preto, SP 14040-903, Brazil
d
Departamento de Biologia, Faculdade de Filosofia, Cieˆncias e Letras de Ribeira˜o 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 (5–25 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 didn’t 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.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Brito et al., 2004). However, despite its toxic effects,
L. camara is extensively used in popular medicine because
Lantana (Lantana camara Linn.) is one of the most of its anti-inflammatory, antipyretic, antispasmodic, and
poisonous weeds in the world. The noxious properties of antibiotic properties (Sharma et al., 2007b).
the plant are well documented: it causes cholestasis, The most well-known lantana compounds are the lan-
hepatotoxicity, photosensitization, and even fatality in tadenes, which belong to the pentacyclic triterpenoid ole-
cattle, horses, sheep, dogs, and humans (Wolfson and anane series. The most abundant triterpene acid is
Solomons, 1964; Tokarnia et al., 1984; Black and Carter, lantadene A (LA); it has been implicated as the main culprit
1985; Fourie et al., 1987; Sharma et al., 1988; Pass, 1991; responsible for the toxic effects of the plant (Sharma et al.,
1991, 2000, 2007b). Despite evidence that mitochondria
* Corresponding author. Tel.: þ55 18 3821 8200; fax: þ55 18 3821 8208. are affected by compounds present in lantana (Sharma
E-mail address: fmingatto@dracena.unesp.br (F.E. Mingatto). et al., 1982; Sharma, 1984), the mechanism by which it

0041-0101/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2010.02.004

particularly in the liver. Plant material v/v) as the eluting solvent (Sharma and Dawra. known as RLA (Fig. Brazil. In the charcoal. Garcia et al. mento de Biologia da Faculdade de Filosofia.. Campus de Dracena. whose COOH major impetus is the membrane potential (Dj) generated H CH3 by electron transport along the respiratory chain in the inner mitochondrial membrane. under this condition. camara and its reduced derivative. 2000 mL of methanol were added and the imental protocols were approved by the Ethical Committee material was macerated for 24 h at room temperature with for the Use of Laboratory Animals of the Faculdade de intermittent shaking. camara Linn. 2007a.5. Merck 7730) using CHCl3-methanol (99. Universidade de Sa˜o Paulo. HO H 2009). The exper- the powder. It is believed that mitochondrial O CH3 H uncoupling is a relevant mechanism for xenobiotic-induced O C C C toxicity. Sa˜o Paulo. 2003). 1).. which yielded a golden yellow extract..3. H3C CH3 To date. membrane potential. v/v) as the mobile phase. 2  40 mL). 1990). 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. 513401000 W). The proton motive force. 1990). 2007b). 1991) to afford the known lantadene A and reduced lantadene A. Animals 2. results from our research group suggest that H CH3 mitochondria are the target organelle of toxic compounds isolated from plants (Mingatto et al.. Ribeira˜o Preto.. no antidote to lantana toxicity is available. The purity of the compounds was imen was identified (number SPFR 10364) by Prof. Cieˆncias e Letras de Ribeira˜o Preto. 1987. Materials and methods lated by thin layer chromatography (silica gel PF254. The influences of lanta- residue (6 g) was chromatographically purified over a silica dene biotransformation on mitochondrial function and gel column (180 g. 2007. 1 mm thickness.1. Milton estimated by thin layer chromatography using different Groppo and deposited in the herbarium of the Departa- solvent systems. 2.5:0. The L. and CHCl3-methanol (99. v/v) and on mitochondrial bioenergetics by assessing their effects extracted with chloroform (CHCl3. Chemical structures of lantadenes used in this study. ATP levels and ATPase layer was dried over anhydrous Na2SO4 and the final dried activity in rat liver mitochondria. which is the major site of H3C CH3 H CH3 xenobiotic uptake and metabolism (Wu et al. 1990).F. The lantadene-rich fraction was further purified and iso- 2. and treatment of its symptoms has met with limited success RLA (Sharma et al. drives ATP synthesis via O H oxidative phosphorylation (Mitchell. as well as 1H NMR analysis. H Mitochondria carry out a variety of biochemical O C C C CH3 processes. we addressed the actions of LA isolated from was removed under reduced pressure.5. A voucher spec- (Sharma et al. 7736) using CHCl3 liver toxicity are considered.5:0.2. mechanism of lantana toxicity at the cellular and molecular levels would help in the development of antidotes and more rational therapies for lantana poisoning. . To 400 g of commercially pelleted rat food (Purina. Santos et al. The organic on respiration. / Toxicon 55 (2010) 1331–1337 induces toxicity has not yet been clearly established. They were fed and ground into powder in an electric grinder. Brazil). it was recently demonstrated that some lan- tadenes exhibit in vitro and in vivo antitumor activity O CH3 (Sharma et al. 2008. but their main function is to produce a majority H3 C CH3 H (>90%) of cellular ATP. Extraction and isolation of lantadenes Male Wistar rats weighing approximately 200 g were housed in plastic cages under regulated temperature The lantana leaf samples were dried in the shade at 37  C (20  C) and the light/dark cycle (12 h:12 h). (Lantana. was suspended in a methanol-H2O mixture (1:7. 60–120 mesh Merck. The extract was filtered through Zootecnia. Universidade Estadual Paulista ‘‘Ju´lio de Mes- a muslin cloth and decolorized with 70 g of activated quita Filho’’. They increase the permeability of the inner mitochondrial membrane to protons along a gradient running from H3 C CH3 intermembrane to matrix spaces. 1961). the organelle is no longer capable of sustaining ATP synthesis (Kadenbach. On the H3 C CH3 other hand. Sa˜o Paulo.. family Verbenaceae) leaf compounds were identified 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. 2008). 2. The solvent present work. an understanding of the Fig. Kaur et al. In COOH addition.1332 A.. Thus. and the residue (15 g) L. Brazil. 1.

1976). pH 7. in a dose-dependent manner.8 (1 mL final a dose-dependent manner. 10 mM HEPES. Garcia et al.5. The chondria energized with either succinate. 65 mM KCl. pH 7.3 mM propranolol in Elvehjem homogenizer. After incubation in the presence of the presence of different concentrations of the lantadenes.4). One milligram of mitochondrial the absence (coupled) and presence (uncoupled) of 1 mM protein was added to 1 mL of respiration buffer containing carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The 2. 5 mM sliced into 50 mL of medium containing 250 mM sucrose. fol- 770  g for 5 min and the resulting supernatant further lowed by 1 mM valinomycin and lantadenes. 7. suspended in 100 mM Tris–HCl. 2000). Pellet was suspended in volume of 1. UK).2 mM EDTA. RLA. / Toxicon 55 (2010) 1331–1337 1333 2. 3. Results 2. but not LA. version 4. The mitochondrial membrane potential (Dj) was esti- mated spectrofluorimetrically using 10 mM safranine O as 2. lantadenes.e. the medium contained consumption was measured using 5 mM succinate 20 mM Tris–HCl (pH 7. and used within 3 h.1.45 mg protein) were incubated in by decapitation and the liver was removed immediately. sodium azide. 0. 0. 2).5 mM EGTA (2 mL final volume).. pH 7. and 10 mM HEPES-KOH. stimulated. CA). ATP quantification 3. Statistical analysis a probe (Zanotti and Azzone. plus 0. 100 mL oxygen after ADP has been exhausted). state-4 respiration of mito- volume). and centrifuged at 15. and centrifuged at a model DU-800 spectrophotometer (Beckman Coulter. a respiratory supernatant was worked-up with a Sigma/Aldrich assay kit chain site II substrate (Fig. and homog.5 mM rotenone) or 5 mM glutamate þ 5 mM malate as addition of 5 mM ATP and stopped by the addition of ice- respiratory substrates in the absence (state-4 respiration) cold 5% trichloroacetic acid. in a final centrifuged at 9800  g for 10 min. Germany). . or glutamate plus malate. and 10 mM HEPES-KOH.000  g for 15 min. (ANOVA) followed by the Dunnett’s test using GraphPad nate (þ2. Scotland. Swelling was 10 mL of medium containing 250 mM sucrose. USA).0 for Windows.8.9. in KOH.7. the mitochondrial suspension (1 mg protein/ The parameters assessed were: state-3 respiration ml) was centrifuged at 9000  g for 5 min at 4  C.5 mM EGTA and 10 mM K2HPO4. state-3 respiration of succinate-energized mitochondria. pH 7. When disrupted mitochondria 7.4. intact (coupled and uncoupled) and in freeze-thawing disrupted mitochondria according to the protocol of 2. CA).5 mL. in 125 mM sucrose. 65 mM KCl. A.2.3 mM estimated from the decrease in absorbance at 540 nm using EGTA. RLA also inhibited. Effects of lantadenes on mitochondrial respiration The amount of ATP was determined using the firefly luciferin–luciferase assay system (Lemasters and Mitochondrial oxygen consumption was monitored in Hackenbrock.4. 1 mM antimycin A. and 0. (2003) with modifications. KOH. Intact mitochondria (1 mg protein/ml) were incubated in a medium containing Mitochondrial respiration was monitored using a Clark. differences were calculated by one-way analysis of variance Mitochondria (2 mg protein) energized with 5 mM succi. 15 mM atrac- enized three times for 15 s at 1-min intervals with a Potter– tyloside. 0. Rats were sacrificed Mitochondria (0. At the concentra- aliquots of the supernatants were neutralized with 5 M tions tested (5–25 mM). 0. at 30  C. CA. 65 mM KCl. pH 7. plus 0.4.1. by measuring the released inorganic phosphate as described by Fiske and Subbarow (1925) at 700 nm using 2.F. The mitochondrial ATPase activity was measured in 1987). and 10 mM HEPES-KOH. and the (consumption of oxygen in the presence of respiratory pellet was treated with 1 mL of ice-cold 1 M HClO4. (San Diego.5 mL (Mingatto et al. in Glasgow. Fullerton.2. 0. 125 mM sucrose. pH type oxygen electrode (Strathkelvin Instruments Limited. and 10 mM HEPES-KOH. mean.000  g for 5 min at 4  C. The reaction was started by the (þ2.4. from GraphPad Software containing 125 mM sucrose. 1978).6. Japan) Data are expressed as mean  s. After substrate and ADP) and state-4 respiration (consumption of centrifugation at 14. Mitochondrial respiration assay Bracht et al. and statistical at the 495/586 nm excitation/emission wavelength pair. Oxygen were used as enzyme source. shown). HEPES-NaOH.. Estimation of mitochondrial membrane potential a model DU-800 spectrophotometer (Beckman Coulter. Pforzheim. pH a final volume of 0. 1980) and a model RF-5301 PC Shimadzu fluorescence spectrophotometer (Tokyo. ATPase activity was evaluated or the presence of 400 nmol ADP (state-3 respiration).1 mM 1 mM EGTA. according to the manufacturer’s instructions and measured which are respiratory chain site I substrates (data not using a SIRIUS Luminometer (Berthold. Homogenate was centrifuged at order to inhibit the inner membrane anion channel. suspended in 1 mL of medium containing 250 mM sucrose and 10 mM HEPES-KOH. and 0. ATPase activity mitochondrial protein concentration was determined by a biuret assay with BSA as the standard (Cain and Skilleter. 4500  g for 15 min.5 mM rotenone) were incubated in a medium Prism.2.1 mM EGTA.10.2 mM EGTA and 5 mM ATP for 20 min at 37  C. pH 7.1% bovine serum albumin. The final mitochondrial pellet was Fullerton. Isolation of rat liver mitochondria 2. a medium containing 54 mM potassium acetate. Mitochondrial swelling in hyposmotic potassium acetate medium Mitochondria were isolated by standard differential centrifugation (Pedersen et al.

The ATPase activity of coupled mitochondria was increased by both LA and RLA at 25 mM. 7 shows that even at 25 mM 3.e. concentration range evaluated. whereas The effects of lantadenes on the ATPase activity were RLA exhibited a significant dose-dependent effect on this measured in intact mitochondria either in the absence parameter along the entire concentration range evaluated. Both compounds didn’t inhibit CCCP-uncoupled respiration indicating that In order to evidence the protonophoric properties of only oxidative phosphorylation was inhibited (Fig. Effects of lantadenes on mitochondrial ATP levels 1. whereas LA was only inhibitory at 25 mM (Fig. Effects of lantadenes on the state-3 respiration rate of succinate- energized rat liver mitochondria. mean of three Materials and methods. Garcia et al.05 and **P < 0. The ATPase activity The effects of lantadenes on mitochondrial ATP levels of uncoupled or disrupted mitochondria was not signifi- were evaluated under the conditions of the respiratory cantly affected by lantadenes.F. circum.5.**Significantly experiments with different mitochondrial preparations.1334 A. 5 shows the effects of lantadenes on Dj of succinate-energized rat liver mitochondria. In accordance 3. *. min-1 mg protein -1) ( nmol O 2 . mean of three experiments with different mitochondrial preparations. as shown in Table 3. / Toxicon 55 (2010) 1331–1337 LA LA 30 75 * 20 50 10 25 (nmol O 2 . (coupled) or in the presence of CCCP (uncoupled) and in freeze-thawing disrupted mitochondria. 2. In agreement with the results on In order to investigate uncoupling of oxidative phos. Under such conditions. compounds (Fig.2. LA phorylation. Fig. Effects of lantadenes on mitochondrial membrane RLA didn’t promote a mitochondrial swelling. we performed mitochondrial swelling in hyposmotic potassium acetate medium. 6). an inhibitor of ATP synthase (FoF1.4. Fig. 4A). a similar pattern was observed for glutamate plus malate (data not assay 15 min after mitochondria were incubated with the shown). mitochondrial respiration and membrane potential. Values represent the mean  s. The assay conditions are described in energized rat liver mitochondria.e. state-4 respiration was induced by addition of only caused a significant decrease in the mitochondrial ATP oligomycin (1 mg/mL).05 and **P < 0. 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 (μM) Lantadene (μM) Fig. Subsequent experiments with CCCP-stimulated mitochondrial respiration were 3. 3). 4B). **Significantly different from control (*P < 0. RLA.01). The assay conditions are described in Materials and methods. Effects of lantadenes on ATPase activity with the results of mitochondrial respiration. Effects of lantadenes on the state-4 respiration rate of succinate- Fig. *. LA only significantly dissipated Dj when present at 25 mM. levels at 25 mM whereas RLA again exhibited a significant ATPase). RLA. different from control (*P < 0.01).3. 3. Effects of lantadenes on mitochondrial swelling performed to test the inhibitor effect of the compounds in in hyposmotic potassium acetate medium the respiratory chain or ATP synthase. vented the oligomycin-imposed inhibition of mitochon- drial state-3 respiration (Fig. but not LA. uncouplers are able to dose-dependent effect on this parameter along the entire increase oxygen consumption. indicating potential (Dj) that RLA is not working as the classical uncouplers like CCCP. Values represent the mean  s. .

Substrates dissolved in the mitochondria aqueous 1998. 2008). believed to be responsible for the toxic effects of lantana chrome e reductase) and then to IV (cytochrome e oxidase).F. 2000). Oligo: oligomycin 1 mg/mL. 1991. exhibited a significant inhibi- The free energy released by this transport is conserved by tory effect on state-3 respiration. which is gradient across the inner mitochondrial membrane. Such a malfunction in stimulating of mitochondrial swelling in hyposmotic mitochondrial bioenergetics instantaneously transforms potassium acetate medium indicates that RLA is acting in mitochondria from cellular powerhouses into a molecular a different way as that expected to occur with a classical .e. 2000. / Toxicon 55 (2010) 1331–1337 1335 A ADP LA 100 ** 75 Oligo 50 25 LA 0 (% of control) RLA or CCCP Δψ RLA CCCP 100 * B 75 ** 50 25 ** KCN ** 0 50 μMO 2 0 5 10 15 20 25 Lantadene (μM) LA or RLA Fig. especially those associated with the liver (Szewczyk and The critical role played by mitochondria in the mainte. LA. it dissipated the mitochondrial membrane potential variety of compounds have been described as uncouplers of and depressed ATP levels at all concentrations in the 5– oxidative phosphorylation (Wallace and Starkov. Amacher. nized. efficiently wasting the metabolic energy of substrates (Wallace and Starkov. Discussion mechanism of several significant toxicities in mammals. Electron transport from involvement of mitochondria in the hepatotoxicity the oxidation of NADH and FADH2 to O2 is tightly coupled to observed in lantana poisoning.. KCN: potassium cyanide 1 mM. 2000).. an ATP synthase increasing the permeability of the inner membrane or inhibitor.. 2002). but only at the highest pumping out protons resulting in an electrochemical Hþ concentration tested. Wojtczak.05 and **P < 0. Garcia et al. Wojtczak. A. which reduce NADþ or FAD. furnace. 2000). (Sharma et al. RLA. from complex I (NADH-coenzyme Q reductase) or II bioenergetics of rat liver mitochondria..01). Effects of lantadenes on the membrane potential of succinate- 1 min energized rat liver mitochondria. Szewczyk and matrix space are oxidized by corresponding dehydroge. *. the lack of and phosphorylation complexes. In addi- process is known as oxidative phosphorylation. 25 mM range. we compared the effects of ATP synthesis. 4. experiments with different mitochondrial preparations. On the other hand. Mitochondrial dysfunction is a fundamental pathogenic 4. The stimulation of state 4 by RLA occurred They decrease the efficiency of ATP production by even in the presence of oligomycin. however. electrochemical potential of this gradient is then harnessed caused a concentration-dependent stimulation of state-4 for the synthesis of ATP by complex V (ATP synthase). this respiration and inhibition of state-3 respiration. mean of three Fig. Thus. Diminishing ATP levels nance of cellular energy metabolism has long been recog. is critical to the development of necrosis (Nicotera et al. The assay conditions are described in Materials and methods. 5. showed that RLA acts as an uncoupler of oxidative decreasing the degree of coupling of the proton motive force phosphorylation (Lu¨de et al. The a biotransformation product of LA (Sharma et al. Values represent the mean  s. Effects of lantadenes (25 mM) on the oligomycin-inhibited state-3 (A) experiments with different mitochondrial preparations. 2002. Figure is representative of three different from control (*P < 0. in order to assess the potential nases. **Significantly and CCCP-uncoupled (1 mM) (B) respiration. A wide tion. 2005). which is (succinate-coenzyme Q reductase) to III (coenzyme cyto. The arrows indicate the addition of the compounds. It occurs through protein-bound redox lantadene A (LA) and reduced lantadene A (RLA) on the centers. 2007b). Wallace and Starkov.

characterized by swelling of hepatocytes.0 * mmol. which is an early lesion in the development turn. A comparison of the structures of LA and RLA. which. The arrow indicates the addition of the in partial fulfillment of the requirements for the Master compounds.99 59. therefore.. individual cell necrosis and biliary ductular hyperplasia (Pass et al.. Values represent the mean  s.17 61. The liver with different mitochondrial preparations.0 LA: lantadene A.0 (coupled and uncoupled) and freeze-thawing disrupted mitochondria with excess of ATP. potassium acetate medium.1336 A. tion. **Significantly different from lesion is consistent with intrahepatic cholestasis and is control (*P < 0.5 indicate that RLA impairs energy metabolism probably due its uncoupler property and suggest that the effect of LA 0.05 and **P < 0. 7. polyuria. 1981). 0 5 10 15 20 25 In line with our results pertaining to RLA. Figure is representative of three experiments with different Universidade Estadual Paulista ‘‘Ju´lio de Mesquita Filho’’. with RLA being that they are not inhibitors of respiratory chain but that more potent than LA. C: control. 1979).F. C or RLA Acknowledgements Δ A 0.80* 35.40  0. probably occurs due a decrease in the Mg2þ- structural feature for the uncoupling activity exhibited by ATPase activity that could be a consequence of ATP deple- RLA. PROPe-Unesp (Programa Primeiros Projetos) and Conselho 1 min Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). Garcia et al. *. when administered to Wistar rats.5 enzyme operates in the reverse direction.71 60. The stimulation of the ATPase activity 5.73 31. which depolarizes the mitochondrial It has been also demonstrated in rats that RLA causes inner membrane through a protonophoric effect (Nicholls. (Pass et al.86  0.0 absence (coupled) or in the presence of CCCP (uncoupled) and in freeze- thawing disrupted mitochondria.01). a condition in which the 7. hydrolyzing ATP (Bracht et al. in general.50  3. 0 5 CCCP This work was supported by grants from Fundaça˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP). that the reduced derivative of lantadene A depresses ATP levels via uncoupling of oxidative phosphorylation.58  1.40 2.e. 7. *P < 0. The inhibition of state-3 respiration by both RLA and LA The present study shows that lantadenes. ATP they are acting as inhibitors of ATP synthase. This constitutes. Results were presented by Andre´a Fontes Fig. mean of three experiments predominantly conjugated bilirubin in the serum. RLA cau- Fig. The assay conditions are described in Materials and constipation.5 Coupled Uncoupled Disrupted mitochondria mitochondria mitochondria 5. suggests that 3-hydroxylation is a determining of cholestasis. mitochondrial preparations. uncoupler as CCCP. Effects of RLA (25 mM) on mitochondrial swelling in hyposmotic Garcia to the Faculdade de Odontologia de Araçatuba. 2003).0 ** in intact coupled mitochondria and the lack of effect in the ** ** ** ATPase activity of uncoupled or disrupted mitochondria 2. in bile canaliculi. 6.97  0. it has been Lantadene (μM) reported that. anorexia. in particular.0 must be related to an effect in other mitochondrial membrane components rather than the enzyme. hepatotoxicity through a toxic metabolite and that injury to 1982).41  3. and accumulation of methods. The assay conditions are described in Materials and methods. RLA: reduced lantadene A.05 relative to control. Effects of lantadenes on the ATP levels in succinate-energized rat ses liver intoxication characterized by jaundice. Brazil.78 ( nmol .5 LA 25 mM 29.84  1. min1 mg protein1 Control 20. It shows. without addition of RLA or CCCP (1 mM). / Toxicon 55 (2010) 1331–1337 LA Table 1 Effect of lantadenes on ATPase activity in intact mitochondria either in the 10.55 RLA 25 mM 30. degree in Cieˆncia Animal.67* 35. dissipate the mitochondrial membrane potential. in turn. polydipsia.64  3. .64  0. mg protein -1) 0.. a potential mechanism of the well- documented lantana toxicity in liver cells. To test this hypothesis we performed experiments to evaluate the RLA effects of lantadenes in the ATPase activity using intact 10. 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. liver mitochondria.

M. Nicholls.D.G...A. The toxicity of diverse Sharma. T... Theory..... H. Salgueiro-Pagadigorria.Z. Acta 1604. Dawra.. 87.B.A. A Bracht. Santos. Pigoso. J. Dehydromonocrotaline Terada. Kra¨henbu¨hl. 2000. Eur. the pentacyclic triterpenoids from lantana (Lantana phosphorylation..R. Pharmacol. Pestana. Med.. 1990. M. B. Rev.L. A. M. Toxicon 20. N.. The authors declare that there are no conflicts of interest. Afr. Pesq. Sharma. T. Sharma.. 136–137. Toxicon detection. Manole. 26. tration. 314. 1991.C. G. V. 54. Sharma. Toxicol... Uncouplers of oxidative phosphorylation..K. Grosso e Rio de Janeiro.).K. O... Mitochondria as a pharmacological Mingatto.E. 120–131. M.. 975–987. Dieterle. 488–493. C.. 2008.. References Santos. J. 1987. 375–400. J. Nat. 932–939.L. do metabolismo em mitocoˆndrias isoladas de tecido animal. and cytotoxicity in isolated rat hepatocytes.. Rev.. Cain. J. Safranine as membrane potential probe in Toxicol.A. Dawra. Am. Cell.K. Oxford. C. Academic Press. Prod. Preparation and caracterization of mitochondria and submitochondrial particles of rat liver and liver- derived tissues. Vet. 2005.. P. Brito. R. Toxicology of Plant between the mitochondrial transmembrane potential. Vet. a switch in Wolfson.P.. Biophys. V. 1984.. ATP concen- and Fungal Compounds. L. Pedersen. 2002...T. Dawra. Skilleter. 29. Santos. (Eds. O. Arch. Bhat. P. Lett.E. Toxicology.. J. Chromat. Biotransformation of Mingatto. Curr. Wojtczak. Poisoning of livestock by lantana plants. Studies on the mechanism of toxicity of reduced lantadene A in rats. Nel. A. Biochem. Environ. C.. Pattabhi. Toxicon 26. Kaur.. Greenawalt. Dorta. (Verbenaceae) em bovinos nos Estados de Mato transfer by a chemiosmotic type of mechanism. Tokarnia. 213–218. Bu¨ter. Intoxicaça˜o Mitchell. J. of the green fruit. plant Lantana camara on guinea pig liver mitochondria. An acute syndrome observed in children following ingestion 139–142. J. Dawra. 153–159.. 515–521.V. 1925.H. Biochem. D. 227–247.). A review of the noxious Hepatocellular toxicity of kava leaf and root extracts.S. M. Bras 4. Mol. as potential antitumor agents. A triterpenoid acid. 33. T. 2003.. Ja¨ggi.C. Peixoto... Biochem. Smith. R. Intracellular ATP. Rev.. Pugh. R.L.. Uyemura. M. Chemopreventive Sharma. 255–265. Lu¨de. Newsholme. pp. In: Snell. M.A. 25–96... Biophys.P. Vet.. S. 2008. S. camara.L. P. J.. D.. Sharma. the pentacyclic triterpenoid phorus. Szewczyk. (Eds. S. C. Sharma. 2000. G. Di Monte.J. Zealand. 1191–1202. Acute 57–63. Biol... C. J. pp.R.B. 131.G.F.. E. inhibits mitochondrial complex I. 37. Rodrigues. reduced lantadene A (22b-angeloyloxyoleanolic acid) in the rat.N. 1985. Crit.. Toxicon 50. Drug-associated mitochondrial toxicity and its mitochondrial permeability transition/cytochrome c release. J.. 2007b.. C. E...J.W. 51. Chem. O. 1981. Silva. Toxicol. D. 1990. F. Mahato. Biochim. Barueri. Biol. Fourie. Toxicol.P. 351–354. Garcia et al. Chem..D. H. 1979. Hackenbrock. Sharma. 411–481. P..A.. R. P. 587..S.. Singh. M. M... Curti. Bioquı´mica. V.. S. 2004. J. 4.B. 1991... 37. Bellomo.B. Nature 191. Lantana camara toxicity in cattle.. D. Bustamente. London. Sharma. 173–178.K. Review of the biochemical effects of Lantana camara toxicological research. A potential mechanism accounting Health Perspect. E. 1154–1160. 2000. F. Makkar. 54.W.. 58.A.. Soper. 1998. rapid kinetics of ATP synthesis in rat liver mitochondria. Carvalho. 1980. Assoc. 1991. Pugh. 12. Toxicol.K. O.. Ishii-Iwamoto. Preparation and use of mitochondria in Sharma. 297–311.J.F. A. J. Biochemical toxicity... for hepatotoxicity of monocrotaline... L. cytotoxicity. Subarrow. Vet. Bras 24. 67.. mitochondria. 1964. Dehydromonocrotaline induces cyclosporine A-insensitive Amacher. target. Lazzari..P. 201. Starkov. aculeata. A. N.). Relationships Anthony.. 1–10. D.P.. Santos. Pharmacol. E. 1984. M. A. 66. R. In: Sharma. S. 217–254.W. J. Methods Cell. Poisoning by fruit of Lantana the decision between apoptosis and necrosis.B. / Toxicon 55 (2010) 1331–1337 1337 Conflict of interest Pass. J. 40..P. P. Thin-layer chromatographic separations Kadenbach. the pentacyclic triterpenoid hepatotoxins of lantana Curti. A. 3961–3962.. 1433–1437. 129–141. pp. D.. C.K. R.. Findlay. C.P. albino mice: AP-1 (c-jun).. H. Mullock. Hullihen. 1222–1227. L. S. and immunohistochemical localization. Molecular structure. 1982. Toxicon 38. Carter.. E.. S. first ed.. M. 2009.A. Black. Leist.. Hum.. Pharmacol. Mitochondrial targets of drug toxicity.. New York.. 30..P.. To¨ro¨k.. Coupling of phosphorylation to electron and hydrogen por Lantana spp. Child.E.. M. Makkar. 107. Nicotera. Biochem..A.. Silva.. and antitumor activity of lantadene-A congeners. K..P. SP. Bansal.P. M. B. Intrinsec and extrinsic uncoupling of oxidative of lantadenes. H. Wu. 1–8.W.. K.P.. K. Uyemura. The colorimetric determination of phos. Effects of nimesulide and its reduced metabolite on plant. 358–362.. morphism and toxicity of lantadene A. 15.W. J.H.. 2008. M. J.D.. 101–127. 1987. Sharma.W. Annu. Y. A. Curti. 6. 77–94. rat liver mitochondria. Isolation and partial activity of lantadenes on two-stage carcinogenesis model in Swiss purification of lantana (Lantana camara L. Lantana poisoning of cattle and sheep in New Sharma. Handbook of Natural Toxins. Synthesis.... Sharma.D. P. B.. Bioenergetics: An Introduction to the Chemiosmotic Wallace. Singh. O estudo Biodivers. O. (Eds. Pharmacol. 102–103. Biochem.. A.A. Arch. Phytochem... A.K. Dorta. R. Van der Lugt.H. Biochem.. 353–388. C. Reynafarje. Dawra. A. Br. pp. Zanotti.P.F. nescence...P. Sharma. Marcel Dekker. 313–352. Mingatto. 1978. Continuous measurement and D from Lantana camara var.. Pass.. D. 2007a. 1982. Phytomedicine plant Lantana camara. 1987.. poly- Fiske. Lantadenes and their esters lantanas for cattle and sheep in Brazil. J.. Tokarnia. Pass. 2003. 1961. Bansal.. Seawright.P. Ramesh. O. Bracht. 173–176.) toxins.B. P. 16–22. Dawra. Maioli.J. 724–730. lantadenes. 144–148. mitoplasts Sharma.P. Pharmacol. S. Findlay.S. I. Toxicol. Pattabhi. Ishii-Iwamoto. Appl. G. Dobereiner.J. Toxicol. M..L. R. O. Sharma... T. S. lantadene Lemasters. camara) plant.L. Vet.. C. Solomons.. 1829–1839. Azzone. F..P.Y. 783–786. 1990.. from the hepatotoxic plant Lantana camara. R. Sharma. IRL Press. S. in guinea pig.. 1988.A. Me´todos de Laborato´rio em review of the hepatotoxic plant Lantana camara.. Bansal. O. Decker. Do¨bereiner. In: Keeler.H. 20.T.. Dis. 1976. H. Makkar. Chem. Biochemical effects of the and inner membrane vesicles determined by firefly-luciferase lumi.. Toxicity of Biophys 282. K. A. Ferrando-May. O. J. 2007. Lett. NFkB (p65) and P53 expression by ELISA 165–172. . M. 71.G. B. A. Pesq. A.E... P.