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0041-0101/$ - se

doi:10.1016/j.to

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Animals of the

number 001 of�CorrespondiE-mail addre

Toxicon 50 (2007) 724–730

www.elsevier.com/locate/toxicon

Dehydromonocrotaline inhibits mitochondrial complexI. A potential mechanism accounting for hepatotoxicity

of monocrotaline$

Fabio E. Mingattoa,�, Daniel J. Dortab, Aline B. dos Santosa, Ivone Carvalhoc,Carlos H.T.P. da Silvac, Vinıcius B. da Silvac, Sergio A. Uyemurad,

Antonio C. dos Santosd, Carlos Curtib

aLaboratorio de Bioquımica, Faculdade de Zootecnia, Universidade Estadual Paulista ‘‘Julio de Mesquita Filho’’, Campus de Dracena,

17900-000 Dracena, SP, BrazilbDepartamento de Fısica e Quımica, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo,

14040-903 Ribeirao Preto, SP, BrazilcDepartamento de Ciencias Farmaceuticas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo,

14040-903 Ribeirao Preto, SP, BrazildDepartamento de Analises Clınicas, Toxicologicas e Bromatologicas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto,

Universidade de Sao Paulo, 14040-903 Ribeirao Preto, SP, Brazil

Received 20 March 2007; received in revised form 5 June 2007; accepted 6 June 2007

Available online 26 June 2007

Abstract

Monocrotaline is a pyrrolizidine alkaloid present in plants of the Crotalaria species, which 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. We evaluated the effects of monocrotaline and its metabolite on respiration, membrane potential and

ATP levels in isolated rat liver mitochondria, and on respiratory chain complex I NADH oxidase activity in submitochondrial

particles. Dehydromonocrotaline, but not the parent compound, showed a concentration-dependent inhibition of glutamate/

malate-supported state 3 respiration (respiratory chain complex I), but did not affect succinate-supported respiration (complex

II). Only dehydromonocrotaline dissipated mitochondrial membrane potential, depleted ATP, and inhibited complex I NADH

oxidase activity (IC50 ¼ 62.06mM) through a non-competitive type of inhibition (KI ¼ 8.1mM). Therefore, dehydromonocrota-

line is an inhibitor of the activity of respiratory chain complex I NADH oxidase, an action potentially accounting for the well-

documented monocrotaline’s hepatotoxicity to animals and humans. The mechanism probably involves change of the complex I

conformation resulting from modification of cysteine thiol groups by the metabolite.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Monocrotaline; Dehydromonocrotaline; Liver mitochondria; Respiratory chain complex I; NADH oxidase activity

e front matter r 2007 Elsevier Ltd. All rights reserved.

xicon.2007.06.006

ement: The experimental protocols used in the work were approved by the Ethical Committee for the Use of Laboratory

Faculdade de Zootecnia, Universidade Estadual Paulista ‘‘Julio de Mesquita Filho’’, Campus de Dracena, protocol

April 3, 2006.

ng author. Tel.: +55 18 3821 8200; fax: +55 18 3821 8208.

ss: fmingatto@dracena.unesp.br (F.E. Mingatto).

ARTICLE IN PRESSF.E. Mingatto et al. / Toxicon 50 (2007) 724–730 725

1. Introduction

Monocrotaline is a pyrrolizidine alkaloid toxinproduced by the Crotalaria genus, which causeshepatotoxic effects in animals and man (Huxtable,1989; Schultze and Roth, 1998). Humans areexposed to monocrotaline following consumptionof herbal teas or contaminated food grains (Huxtable,1989); animals are exposed following ingestion ofcontaminated ration components like corn, soybeanor sorghum (Souza et al., 1997). Monocrotaline’stoxicity requires bio-activation by liver cytochromeP-450 to dehydromonocrotaline (Butler et al., 1970;Lafranconi and Huxtable, 1984; Wilson et al., 1992;Pan et al., 1993; Schultze and Roth, 1998), a highlyreactive bifunctional alkylating agent that binds tocellular DNA and proteins (Petry et al., 1984;Hincks et al., 1991; Niwa et al., 1991; Wagner et al.,1993; Yan and Huxtable, 1995). Dehydromonocro-taline appears to be detoxified by conjugation withglutathione (GSH) (Yan and Huxtable, 1995).Accordingly, sulphur-containing amino acids likecysteine and taurine prevent against the toxic effectsof monocrotaline (Hayashi and Lalich, 1968; Yanand Huxtable, 1996).

Oxidation of energy-linked substrates in mito-chondria produces reducing equivalents, which aretransferred via pyridine (NADH) or flavine (FADH2)nucleotides to the respiratory chain. A protonelectrochemical gradient having membrane poten-tial as the major component, capable of sustainingATP synthesis, is then generated (Mitchell, 1961;Nicholls, 1982). It is worth to recall in thisconnection that mitochondria have often beenconsidered an important target of xenobiotics,which may cause cell injury via ATP depletion(Wallace and Starkov, 2000).

Complex I provides the site of entry of electronsfrom NADH to the mitochondrial electron trans-port chain. Mammalian complex I is a very largeenzyme, containing 46 different subunits with atotal molecular mass of �980 kDa (Caroll et al.,2003); its supramolecular organization and struc-tural relation to the remainder of the respiratorychain, however, still remains unknown (Lenaz et al.,2006). The electrons’ transfer from NADH (oxidation)via complex I reduces ubiquinone (coenzyme Q),generating part of the electrochemical gradientrequired for ATP synthesis. Therefore, inhibitionof respiratory chain complex I could constitute animportant mechanism of cell ATP depletion and,therefore, of cell death. In the present study, we

demonstrate that dehydromonocrotaline is an in-hibitor of NADH oxidase activity of respiratorychain complex I, an action potentially accountingfor the well-documented monocrotaline’s hepato-toxicity to animals and humans.

2. Materials and methods

2.1. Chemicals

Monocrotaline was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dehydromono-crotaline was prepared from monocrotaline accord-ing to Mattocks et al. (1989). The purity of theresulting pyrrole was confirmed using NMR. Allother reagents were of the highest commerciallyavailable grade. Dimethyl sulphoxide used fordissolving monocrotaline and dehydromonocrota-line had no effect on the assays. All stock solutionswere prepared using glass-distilled deionized water.

2.2. Animals

Male Wistar rats weighing approximately 200 gwere used in this study. Animals were maintained ata maximum of four rats per cage under standardlaboratory conditions, and water and food weregiven ad libitum. The experimental protocols wereapproved by the Ethical Committee for the Use ofLaboratory Animals of the Faculdade de Zootecnia,Universidade Estadual Paulista ‘‘Julio de MesquitaFilho’’, Campus de Dracena.

2.3. Isolation of rat liver mitochondria

Mitochondria were isolated by standard differ-ential centrifugation (Pedersen et al., 1978). MaleWistar rats weighing approximately 200 g weresacrificed by cervical dislocation; livers (10–15 g)were immediately removed, sliced in 50ml ofmedium containing 250mM sucrose, 1mM EGTAand 10mM HEPES-KOH, pH 7.2, and homoge-nized three times for 15 s at 1min intervals with aPotter-Elvehjem homogenizer. Homogenates werecentrifuged at 580g for 5min and the resultingsupernatant was further centrifuged at 10,300g for10min. Pellets were suspended in 10ml of mediumcontaining 250mM sucrose, 0.3mM EGTA, and10mM HEPES-KOH, pH 7.2, and centrifuged at3400g for 15min. The final mitochondrial pellet wassuspended in 1ml of medium containing 250mMsucrose and 10mMHEPES-KOH, pH 7.2, and used

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within 3 h. Mitochondrial protein content wasdetermined by the biuret reaction.

2.4. Preparation of submitochondrial particles

For preparation of submitochondrial particles,the mitochondrial pellet was frozen and stored at�20 1C. After 24 h, it was thawed and suspendedwith the homogenization medium to 20mg protein/ml. The mitochondrial suspension was subjected fivetimes to 10 s of sonic oscillation with 30 s intervalsusing 80W. The suspension was centrifuged at9750g for 10min, and the submitochondrial parti-cles in the supernatant were isolated by centrifuga-tion at 100,000g during 1 h and stored at �70 1C(Uyemura and Curti, 1992).

2.5. Mitochondrial respiration assay

Mitochondrial respiration was determined bymonitoring oxygen consumption using a Clark-typeoxygen electrode (Strathkelvin Instruments Limited,Glasgow, Scotland, UK). One milligram of mito-chondrial protein was added to 1ml of standardmedium containing 125mM sucrose, 65mM KCl,and 10mM HEPES-KOH, pH 7.4, plus 0.5mMEGTA and 10mM K2HPO4, at 30 1C. The oxidiz-able substrates used were 5mM glutamate+5mMmalate or 5mM succinate (+ 2.5 mM rotenone), inthe absence (basal respiration) or presence (state 3respiration) of 400 nmol ADP.

2.6. Estimation of mitochondrial membrane potential

The mitochondrial membrane potential wasmonitored spectrofluorimetrically using 10 mM sa-franine O as a probe (Zanotti and Azzone, 1980)and a Model F-4500 Hitachi fluorescence spectro-photometer (Tokyo, Japan) at the 495/586 nmexcitation/emission wavelength pair. Mitochondria(2mg protein) were incubated in the standardmedium described above, plus 0.5mM EGTA(2ml final volume).

2.7. ATP determination

ATP was determined by the firefly luciferin–luci-ferase assay system (Lemasters and Hackenbrock,1976). The mitochondrial suspension (1mg protein/ml) was centrifuged at 9000g for 5min at 4 1C andthe pellet treated with 1ml of ice-cold 1M HClO4.After centrifugation at 14,000g for 5min at 4 1C,

100 ml aliquots of the supernatants were neutralizedwith 70 ml of 2M KOH, suspended in 100mMTRIS–HCl, pH 7.8 (final volume, 1ml), andcentrifuged at 15,000g for 15min. Bioluminescencewas measured in the supernatant with a Sigma-Aldrich assay kit, according to the manufacturer’sinstructions, using an AutoLumat LB953 lumines-cence photometer (Perkin-Elmer Life Sciences,Wilbad, Germany).

2.8. NADH oxidase activity assay

NADH oxidase activity was assayed at 30 1C insubmitochondrial particles, as decrease of absor-bance at 340 nm employing NADH as substrate.The standard assay mixture contained 125mMsucrose, 65mM KCl and 10mM HEPES-KOH,pH 7.4, plus 0.2mM EGTA. The enzymaticreaction was started with submitochondrial particles(10 mg protein/ml). The kinetic parameters Vmax

(maximum velocity), Km (Michaelis constant) andKI (inhibition constant), were calculated using theSigraf software (Leone et al., 2005).

2.9. Statistical analysis

Data were expressed as mean7SD and statisticaldifferences were calculated by one-way analysis ofvariance followed by the Dunnett’s test using theGraphPad Prism software, version 4.0 for Win-dows, GraphPad Software (San Diego, CA, USA).Values of Po0.05 were considered significant(Fig. 1).

3. Results

3.1. Effects of monocrotaline and

dehydromonocrotaline on respiration, membrane

potential and ATP levels of mitochondria

Oxygen consumption was monitored in mito-chondria exposed to 1–500 mM monocrotaline ordehydromonocrotaline, in order to evaluate state 3(ADP-stimulated) or state 4 (basal) respiration ofthe organelles energized either by the NAD+-linkedsite I substrates glutamate+malate or by the FAD-linked site II substrate succinate (+ rotenone).Monocrotaline did not significantly affect respirationof mitochondria energized with either substrates(results not shown), but dehydromonocrotalinesignificantly inhibited the glutamate/malate-supportedstate 3 respiration in a dose-dependent manner

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N

OO

O

O

OH OH

CH3

CH3 CH3

N

OO

O

O

OH OH

CH3

CH3 CH3

MCT DHM

Fig. 1. Chemical structures of monocrotaline (MCT) and

dehydromonocrotaline (DHM).0 50 100 150 200 250

0

10

20

30

40

**

*

*

Ox

yg

en

co

ns

um

pti

on

(nm

ol O

2/m

in/m

g p

rote

in)

0 50 100 150 200 250

0

25

50

75

100

**

**

Δψ(%

of

co

ntr

ol)

0 50 100 150 200 250

0.0

2.5

5.0

7.5

10.0

*

*

*

*

Dehydromonocrotaline ( μM)

AT

P

(nm

ol/

mg

pro

tein

)

Fig. 2. Concentration–response curves for the effects of dehy-

dromonocrotaline on state 3 respiration (A), membrane potential

(B), and ATP levels (C) in glutamate/malate-energized isolated

rat liver mitochondria incubated in the standard medium

(125mM sucrose, 65mM KCl, and 10mM HEPES-KOH, pH

7.4) as described in Materials and methods. Data are mean7SD

of three independent experiments. *Significantly different from

controls (absence of dehydromonocrotaline) (Po0.05).

F.E. Mingatto et al. / Toxicon 50 (2007) 724–730 727

(between 100 and 250mM) (Fig. 2A). This resultindicates that dehydromonocrotaline, but not mono-crotaline, is a respiratory chain complex I inhibitor.Accordingly, while monocrotaline did not altermitochondrial membrane potential in glutamate/ma-late-energized mitochondria (result not shown), dehy-dromonocrotaline caused its dissipation (Fig. 2B).Moreover, the inhibition of state 3 respiration and thedissipation of membrane potential of mitochondria bydehydromonocrotaline were accompanied by a sig-nificant mitochondrial ATP depletion (Fig. 2C).

3.2. Effect of dehydromonocrotaline on complex I

NADH oxidase activity of submitochondrial particles

The effect of dehydromonocrotaline on NADHoxidase activity of respiratory chain complex I wasevaluated in submitochondrial particles employing50 mM NADH as substrate, in the absence ofexogenously added electron acceptor. The inhibitionof this activity by 1 mM rotenone (result not shown)indicates that it indeed is associated with complex I.Initial velocities (V0) were calculated from the slopesof the lines representing NADH consumption bythe enzyme at the beginning of the activity curves(not shown). The progress of the concentration–re-sponse curve, as well as its respective IC50 value, isshown in Fig. 3. The type of inhibition ofdehydromonocrotaline and respective KI weredetermined from the double-reciprocal plot at aconcentration corresponding to its IC50 value(Fig. 4). Dehydromonocrotaline presented a non-competitive type of inhibition in relation to thebinding site of NADH, and a KI value of 8.1 mM.

4. Discussion

Monocrotaline, an alkaloid pyrrolizidine phyto-toxin, has well-documented hepatotoxicity for both

animals and man (Mclean, 1970; Mattocks, 1986;Huxtable, 1989; Stegelmeier et al., 1999; Nobreet al., 2004). In the liver, monocrotaline causescentrilobular cell necrosis, dilated, congested sinu-soids, hemorrhage, injury to central venous andsinusoid endothelial cells (Deleve et al., 1999; Yee

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0 50 100 150 200 250 300

0

25

50

75

100

IC50

=62.06 μM

Dehydromonocrotaline (μM)

NA

DH

oxid

ase a

cti

vit

y(%

of

inh

ibit

ion

)

Fig. 3. Concentration–response curve for the effects of dehy-

dromonocrotaline on submitochondrial particles (10mg protein)

NADH oxidase activity employing 50 mMNADH as substrate, at

30 1C, in the standard medium described in legend of Fig. 2, in the

presence of 0.2mM EGTA (1ml final volume). Data are

mean7SD of three independent experiments. Percent of inhibi-

tion in relation to control (absence of dehydromonocrotaline):

V0 ¼ 2.570.2 nmol NADH/min/mg protein.

-0.10 -0.05 0.00 0.05 0.10 0.15 0.20

0.5

1.0KI = 8.1 μM

1/NADH (1/μM)

1/V

0(1

/nm

ol/

min

/mg

pro

tein

)

Fig. 4. Double-reciprocal plot for the inhibition of submitochon-

drial particles NADH oxidase activity by dehydromonocrotaline,

assayed as described in the legend of Fig. 3. Data are mean7SD

of three independent experiments. (J) Absence of dehydromo-

nocrotaline; (K) presence of dehydromonocrotaline in concen-

tration corresponding to the IC50 value (62.06mM). Km and Vmax

for control (absence of dehydromonocrotaline): 12.35mM and

24.38 nmol NADH/min/mg protein, respectively. KI ¼ inhibition

constant.

F.E. Mingatto et al. / Toxicon 50 (2007) 724–730728

et al., 2000; Copple et al., 2002), and parenchymacell oncosis and apoptosis (Copple et al., 2004). Thecytochrome P-450 in the liver bio-activates mono-crotaline to an alkylating pyrrole derivative, thedehydromonocrotaline, considered to be responsi-ble for its toxic effects (Butler et al., 1970;Lafranconi and Huxtable, 1984; Roth and Reindel,1990; Pan et al., 1993).

Mitochondrial dysfunction is a relevant mechan-ism for the pathogenesis of several importanttoxicities in mammals, particularly in the liver(Amacher, 2005). In order to assess a potentialinvolvement of mitochondria on monocrotalinehepatotoxicity, we addressed its effects, as well asthe effects of its metabolite dehydromonocrotaline,on energy-linked parameters of isolated rat liverorganelles. Monocrotaline did not affect mitochondrialrespiration, membrane potential, or ATP levels,while dehydromonocrotaline caused concentration-dependent inhibition of glutamate/malate-sup-ported state 3 respiration, dissipation of membranepotential and ATP depletion in mitochondria, aswell as concentration-dependent inhibition of com-plex I NADH oxidase activity in submitochondrialparticles.

Although respiratory chain complex I is a well-established molecular target for several natural andsynthetic compounds (Degli Esposti, 1998; Lum-men, 1998; Miyoshi, 1998; Granell et al., 2004), themechanisms underlying their interactions are littleunderstood. The non-competitive inhibition ofdehydromonocrotaline indicates that it binds tocomplex I at another site than its NADH-bindingsite, possibly inducing a change in the shape ofmolecule and hindering NADH oxidation. Indeed,flexible docking calculations performed in order topropose a binding mode of these compounds withthe complex I subunit-B8, one of the mostconserved complex I subunit with apparent regula-tory function (Brockmann et al., 2004), showed thatthey may interact with groups other than thoseinvolved in a potential active site; interaction ofdehydromonocrotaline appeared more favourablethan interaction of monocrotaline (results notshown). However, dehydromonocrotaline is analkylating agent that may bind to thiol groups ofmolecules like GSH or proteins (Yan and Huxtable,1996). Accordingly, we have observed that exposureof isolated rat liver mitochondria to dehydromono-crotaline at concentrations inhibiting complex Iactivity (100–250 mM) resulted in significant oxida-tion of GSH (result not shown). It has beenreported that the activity of complex I is regulatedby thiol groups (Shivakumar et al., 1995; Balijepalliet al., 1999), and that reversible oxidation ofcomplex I cysteine thiols causes its inhibition (Jhaet al., 2000). Indeed, complex I presents severalcysteine residues (Schilling et al., 2005), whosemodification would influence its conformation andfunction. Therefore, it is reasonable to propose a

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role of thiol modification in the dehydromonocrota-line-induced inhibition of complex I NADH oxidaseactivity. The consequences of this action are thesequential impairments of respiratory chain ubiqui-none reduction, translocation of protons across themitochondrial membrane, generation of the electro-chemical gradient, and ATP synthesis.

It has been well documented that the decrease ofATP levels, mainly due to inhibition of itsmitochondrial synthesis, is a critical event causingcell death by necrosis (Nicotera et al., 1998; Wallaceand Starkov, 2000; Szewczyk and Wojtczak, 2002).Our results suggest that such an effect, caused byinhibition of NADH oxidase activity of respiratorychain complex I by dehydromonocrotaline, ispotentially a mechanism accounting for the well-documented monocrotaline’s hepatotoxicity to ani-mals and humans, probably via change of thecomplex I conformation resulting from modificationof cysteine thiol groups by the metabolite.

Acknowledgements

This work was supported by grants from Fundac-aode Amparo a Pesquisa do Estado de Sao Paulo(FAPESP), Process number 04/09882-7, and Con-selho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq), Brazil.

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