Toxicology in Vitro 26 (2012) 51–56 Contents lists available at SciVerse ScienceDirect Toxicology in Vitro journal homepage: www.elsevier .com/locate / toxinvi t Abamectin affects the bioenergetics of liver mitochondria: A potential mechanism of hepatotoxicity Juliana C. Castanha Zanoli, Marcos A. Maioli, Hyllana C.D. Medeiros, Fábio E. Mingatto ⇑ Laboratório de Bioquímica Metabólica e Toxicológica (LaBMeT), UNESP – Univ Estadual Paulista, Campus de Dracena, 17900-000 Dracena, SP, Brazil a r t i c l e i n f o Article history: Received 28 August 2011 Accepted 8 October 2011 Available online 17 October 2011 Keywords: Abamectin Mitochondria FoF1-ATPase Oxidative phosphorylation Adenine nucleotide translocator ATP synthesis 0887-2333/$ - see front matter � 2011 Elsevier Ltd. A doi:10.1016/j.tiv.2011.10.007 ⇑ Corresponding author. Tel.: +55 18 3821 8158; fa E-mail address: fmingatto@dracena.unesp.br (F.E. a b s t r a c t Abamectin (ABA) is a macrocyclic lactone of the avermectin family used worldwide as an antiparasitic agent in farm animals and pets and as the active ingredient of insecticides and nematicides. In this study, the effects of abamectin on the bioenergetics of mitochondria isolated from rat liver were evaluated. Mitochondria are responsible for converting the energy released by electron transport and stored as the binding energy molecule ATP. Xenobiotics that interfere with its synthesis or utilization can be acutely or chronically toxic. Abamectin (5–25 lM) caused concentration-dependent inhibition of the respiratory chain without affecting the membrane potential or the activity of enzymes NADH dehydro- genase or succinate dehydrogenase. This behavior is similar to oligomycin and carboxyatractyloside and suggests direct action on FoF1-ATPase and/or the adenine nucleotide translocator (ANT). ABA more pronouncedly inhibited ATPase phosphohydrolase activity in intact, uncoupled mitochondria than in freeze–thawed disrupted mitochondria. ADP-stimulated depolarization of the mitochondrial membrane potential was also inhibited by ABA. Our results indicate that ABA interacts more specifically with the ANT, resulting in functional inhibition of the translocator with consequent impairment of mitochondrial bioenergetics. This effect could be involved in the ABA toxicity to hepatocytes. � 2011 Elsevier Ltd. All rights reserved. 1. Introduction Abamectin (ABA) is obtained by natural fermentation of Strepto- myces avermitilis, which provides a mixture of avermectins consist- ing of P80% of avermectin B1a and 620% avermectin B1b (Agarwal, 1998). B1a and B1b (Fig. 1) have similar biological and toxicological properties (Hayes and Laws, 1990). Abamectin is currently used in several countries as a pest control agent in livestock and as an ac- tive principle of nematicides and insecticides for agricultural use (Kolar et al., 2008). ABA is highly toxic to insects and may be highly toxic to mammals (Lankas and Gordon, 1989). Seixas et al. (2006) reported that ABA poisoning caused the death of 57 calves over 4 years. The authors noted that this number, caused by incorrect dosage to the animals, might be underestimated because signs of intoxication vary in intensity and many animals recover quickly. Despite its restricted use to animals and crops, several cases of accidental or intentional abamectin poisoning in human also have been described (Chung et al., 1999; Yang, 2008). Due to its interposition between the digestive tract and the gen- eral circulation of the body, the liver has an important role in metabolism and biotransformation of exogenous substances. Therefore, it receives large amounts of nutrients and xenobiotics ll rights reserved. x: +55 18 3821 8208. Mingatto). absorbed through the digestive tract and portal vein, becoming the target organ of several classes of toxicants and natural or syn- thetic toxins (Guillouzo, 1998). The most direct mechanism of liver toxicity, at the cellular and molecular level, is the specific interac- tion of the toxicant with a critical cellular component (mitochon- dria, for example) and subsequent modulation of its function (Meyer and Kulkarni, 2001). ABA poisoning can impair the function of hepatocytes. Research conducted by Hsu et al. (2001) showed elevated levels of the en- zyme aspartate aminotransferase (AST) in the blood serum of rats after exposure to ABA by gavage at doses between 1 and 20 mg/kg body weight. The maximum activity was obtained with a dose of 20 mg/kg of body weight 1 h after ingestion. Eissa and Zidan (2010), using a commercial product, also observed signs of aba- mectin liver toxicity, with increased activity of the enzyme AST in rats treated with doses equivalent to 1/10 or 1/100 of the LD50 (18 mg/kg) in the diet of animals over 30 consecutive days. In addi- tion, El-Shenawy (2010) undertook a comparative study of the in vitro toxic action of some insecticides, including ABA at concen- trations of 10 and 100 lM, on isolated rat hepatocytes. There was a significant increase in alanine aminotransferase (ALT) and aspar- tate aminotransferase (AST) activity when hepatocytes were incu- bated for 30 min with either concentration of ABA. This activity persisted after 120 min, the longest time point for which data was collected. http://dx.doi.org/10.1016/j.tiv.2011.10.007 mailto:fmingatto@dracena.unesp.br http://dx.doi.org/10.1016/j.tiv.2011.10.007 http://www.sciencedirect.com/science/journal/08872333 http://www.elsevier.com/locate/toxinvit Fig. 1. Chemical structures of abamectin components avermectin B1a and B1b. 52 J.C. Castanha Zanoli et al. / Toxicology in Vitro 26 (2012) 51–56 Mitochondria carry out a variety of biochemical processes, but their main function is to produce a majority (>90%) of cellular ATP. The proton motive force, whose major impetus is the mem- brane potential (Dw) generated by electron transport along the respiratory chain in the inner mitochondrial membrane, drives ATP synthesis via oxidative phosphorylation (Mitchell, 1961). Experimental evidence from our research group indicates that mitochondria represent a primary target critical for the action of drugs and toxins (Mingatto et al., 2000, 2007; Garcia et al., 2010). Here, we addressed the actions of ABA on mitochondrial bioenergetics by assessing its effect on respiration, membrane po- tential, ATP levels, activity of mitochondrial respiratory chain en- zymes, ATPase and ANT in isolated rat liver mitochondria. 2. Materials and methods 2.1. Chemicals Abamectin, containing 92% avermectin B1a and 8% avermectin B1b, was kindly supplied by the company Ourofino Agribusiness (Cravinhos, São Paulo, Brazil). All other reagents were of the high- est commercially available grade. Dimethyl sulfoxide (DMSO) used to dissolve abamectin had no effect on the assays. The volume of DMSO added never exceeded 0.1% of the total volume of medium. All stock solutions were prepared using glass-distilled deionized water. 2.2. Animals Male Wistar rats weighing approximately 200 g were used in this study. The animals, provenient from the Central Bioterium of the São Paulo State University, Botucatu, SP, Brazil, were main- tained with a maximum of four rats per cage under standard labo- ratory conditions, while water and food were provided ad libitum. The experimental protocols were approved by the Ethical Commit- tee for the Use of Laboratory Animals of the Universidade Estadual Paulista ‘‘Júlio de Mesquita Filho’’, Campus de Dracena. 2.3. Isolation of intact and disrupted rat liver mitochondria Mitochondria were isolated by standard differential centrifuga- tion (Pedersen et al., 1978). Rats were sacrificed by decapitation, and the liver was immediately removed, sliced into 50 ml of med- ium containing 250 mM sucrose, 1 mM EGTA, and 10 mM HEPES- KOH, pH 7.2, and homogenized three times for 15 s at 1-min inter- vals with a Potter-Elvehjem homogenizer. Homogenate was centri- fuged at 770g for 5 min, and the resulting supernatant further centrifuged at 9800g for 10 min. The 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 4500g for 15 min. The final mitochondrial pellet was suspended in 1 ml of medium containing 250 mM sucrose and 10 mM HEPES-KOH, pH 7.2 and was used within 3 h. The mitochondrial protein concentration was determined by a biuret assay with BSA as the standard (Cain and Skilleter, 1987). The disrupted mitochondria were obtained by heat shock treat- ment after three consecutive cycles of freezing in liquid nitrogen and thawing in a water bath heated to 37 �C. The membrane frag- ments were kept at 4 �C and were used in the assessment of mito- chondrial enzymatic activity within 3 h. 2.4. Mitochondrial respiration assay Mitochondrial respiration was monitored using a Clark-type oxygen electrode (Strathkelvin Instruments Limited, Glasgow, Scotland, UK), and respiratory parameters were determined according to Chance and Williams (1955). One milligram of mito- chondrial protein was added to 1 ml of respiration buffer contain- ing 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 consump- tion was measured using 5 mM glutamate + 5 mM malate, 5 mM succinate (+2.5 lM rotenone) or 200 lM N,N,N,N-tetramethyl-p- phenylene diamine (TMPD) + 3 mM ascorbate as respiratory sub- strates in the absence (state-4 respiration) or the presence of 400 nmol ADP (state-3 respiration). 2.5. Estimation of mitochondrial membrane potential (Dw) The mitochondrial membrane potential (Dw) was estimated spectrofluorimetrically using model RF-5301 PC Shimadzu fluores- cence spectrophotometer (Tokyo, Japan) at the 495/586 nm excita- tion/emission wavelength pair. Safranine O (10 lM) was used as a probe (Zanotti and Azzone, 1980). Mitochondria (2 mg protein) energized with 5 mM glutamate + 5 mM malate 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 final volume). 2.6. ATP quantification ATP levels were determined using the firefly luciferin–luciferase assay system (Lemasters and Hackenbrock, 1976). After incubation in the presence of ABA, the mitochondrial suspension (1 mg pro- tein/ml) was centrifuged at 9000g for 5 min at 4 �C, and the pellet was treated with 1 ml of ice-cold 1 M HClO4. After centrifugation at 14000g for 5 min at 4 �C, 100 ll aliquots of the supernatants were neutralized with 5 M KOH, suspended in 100 mM TRIS–HCl, pH 7.8 (1 ml final volume), and centrifuged at 15000g for 15 min. The supernatant was worked up with a Sigma/Aldrich assay kit (Cata- log Number FLAA) according to the manufacturer’s instructions and measured using a SIRIUS Luminometer (Berthold, Pforzheim, Germany). 2.7. Mitochondrial ATPase activity Mitochondrial ATPase activity was measured in intact-uncou- pled and freeze–thawing-disrupted mitochondria according to the protocol of Bracht et al. (2003), with modifications. Intact mito- chondria (1 mg protein/ml) were incubated in a medium contain- 0 10 20 30 ** **** ** O xy ge n co ns um pt io n (n m ol O 2 . m in -1 . m g pr ot ei n -1 ) 0 5 10 15 20 25 0 25 50 75 ** **** ** Abamectin (μM) O xy ge n co ns um pt io n (n m ol O 2 . m in -1 . m g pr ot ei n -1 ) A B Fig. 2. Effect of abamectin on the state-3 respiration rate of glutamate plus malate (A) and succinate-energized (B) rat liver mitochondria. Assay conditions are described in Section 2. Values represent the mean ± S.E. mean of three experiments with different mitochondrial preparations. ⁄⁄Significantly different from control (P < 0.01). CCCP J.C. Castanha Zanoli et al. / Toxicology in Vitro 26 (2012) 51–56 53 ing 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 pres- ence of 1 lM carbonyl cyanide m-chlorophenyl hydrazone (CCCP), in a final volume of 0.5 ml. When disrupted mitochondria were used as the enzyme source, the medium contained 20 mM TRIS– HCl (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 released inorganic phosphate, as described by Fiske and Subbarow (1925), at 700 nm using a DU-800 spectrophotometer (Beckman Coulter, Ful- lerton, CA). Results were expressed as nmol Pi. min�1. mg protein�1. Sensi- tivity to oligomycin (1 lg/ml) was tested in all mitochondrial suspensions. 2.8. Determination of enzyme activity related to mitochondrial respiratory chain (NADH and succinate dehydrogenase) The activity of NADH and succinate dehydrogenases was mea- sured spectrophotometrically according to Bracht et al. (2003), using a DU-800 spectrophotometer (Beckman Coulter, Fullerton, CA). The reaction medium (final volume 1.5 ml) contained 20 mM TRIS, pH 7.4, and 1 lM Antimycin A. Disrupted mitochon- dria (0.2 mg/ml) were added along with one of four abamectin con- centrations (5, 10, 15 and 25 lM), either 1 mM NADH or 10 mM succinate, and 0.4 mM potassium ferricyanide as electron acceptor. The amount of ferricyanide reduced was determined by the de- crease in absorbance at 420 nm and enzyme activity was repre- sented as nmol. min�1. mg protein�1, using 1.04 mM�1 as the molar extinction coefficient of ferricyanide. 2.9. Inhibition of ADP-induced depolarization of Dw Inhibition of ADP-induced depolarization of Dw was performed as described (O’Brien et al., 2008) with modifications. Freshly iso- lated mitochondria were pre-incubated in the presence of 5– 25 lM ABA or 5 lM carboxyatractyloside (cATR) and then ener- gized with 5 mM succinate for 1.5 min before adding 400 nmol ADP. ADP-induced depolarization describes the change and recov- ery in Dw upon addition of ADP. The amplitude of depolarization induced by ADP was measured in the presence and absence of the test compounds. 2.10. Statistical analysis Data are expressed as the mean ± S.E. mean, and statistical dif- ferences were calculated using one-way analysis of variance (ANO- VA) followed by the Dunnett́s test using GraphPad Prism, v 4.0 for Windows (GraphPad Software, San Diego, CA, USA). 1 min 50 μm ol O 2 Oligo, cATR or ABA KCN Fig. 3. Effect of abamectin (ABA, 25 lM) on CCCP-uncoupled (1 lM) respiration. The figure is representative of three experiments with different mitochondrial preparations. Arrows indicate addition of compounds. Oligo: oligomycin 1 lg/ml. cATR: carboxyatractyloside 1 lM. KCN: potassium cyanide 1 lM. RFI: relative fluorescence intensity. 3. Results 3.1. Effects of abamectin on mitochondrial respiration Mitochondrial oxygen consumption was monitored in the pres- ence of varying concentrations of ABA. The parameters assessed were state-3 respiration (consumption of oxygen in the presence of respiratory substrate and ADP) and state-4 respiration (con- sumption of oxygen after ADP has been exhausted). At the concen- trations tested (5–25 lM), ABA inhibited state-3 respiration of mitochondria in a concentration-dependent manner. This effect was observed when mitochondria were energized with either glu- tamate plus malate, the respiratory chain site I substrates (Fig. 2A), or succinate, a respiratory chain site II substrate (Fig. 2B). A maxi- mum effect was observed at a concentration of 15 lM. ABA also inhibited state-3 respiration of TMPD plus ascorbate-energized mitochondria in a concentration-dependent manner (data not shown). The compound did not stimulate state-4 respiration, indi- cating that it does not act as an uncoupler (data not shown). Subsequent experiments with carbonyl cyanide m-chloro- phenyl hydrazone (CCCP)-stimulated mitochondrial respiration were performed to test the inhibitor effect of the compound on the respiratory chain or on ATP synthase. ABA did not inhibit CCCP-uncoupled respiration, indicating that only oxidative phos- phorylation was inhibited (Fig. 3). The same behavior was ob- C Olig o 5 10 15 25 0 1 2 3 4 5 6 Abamectin (μM) ** ** **** AT P (n m ol . m g pr ot ei n -1 ) Fig. 5. Effect of abamectin (ABA) on the ATP levels in glutamate plus malate- energized rat liver mitochondria. Assay conditions are described in Section 2. Values represent the mean ± S.E. mean of three experiments with different mitochondrial preparations. C: control, only 0.1% DMSO. Oligo: oligomycin 1 lg/ ml. ⁄⁄Significantly different from control (P < 0.01). 100 150 * e ac tiv ity -1 . m g pr ot ei n -1 ) A 54 J.C. Castanha Zanoli et al. / Toxicology in Vitro 26 (2012) 51–56 served with oligomycin (ATPase inhibitor) and carboxyatractylo- side (ANT inhibitor). 3.2. Effect of abamectin on mitochondrial membrane potential (Dw) Figure 4 shows the effect of ABA on the Dw of gluta- mate + malate-energized rat liver mitochondria. ABA (25 lM) did not dissipate Dw. The same behavior was observed for oligomycin and carboxyatractyloside. At the end of the experiment, 1 lM CCCP (uncoupler) or 2.5 lM rotenone (complex I inhibitor) was added as a positive control, and the mitochondrial membrane electrical po- tential dissipated. 3.3. Effect of abamectin on mitochondrial ATP levels The effect of ABA on mitochondrial ATP levels was evaluated using the respiratory assay conditions 15 min after mitochondria were incubated with the compound (Fig. 5). In agreement with the mitochondrial respiration results, ABA caused a significant con- centration-dependent decrease in mitochondrial ATP levels, reach- ing a maximum effect at 15 lM. 3.4. Effects of abamectin on the FoF1-ATPase activity The effects of ABA on FoF1-ATPase activity were measured in in- tact-uncoupled mitochondria in the presence of CCCP, and in freeze–thawing-disrupted mitochondria, as shown in Fig. 6A and B, respectively. The ATPase activity of uncoupled mitochondria was increased in a concentration-dependent manner by ABA (Fig. 6A). In disrupted mitochondria, the effects were less dramatic and similar across all concentrations tested (Fig. 6B). 3.5. Effect of abamectin on NADH and succinate dehydrogenase activities The effect of ABA on NADH and succinate dehydrogenase activ- ity was measured in freeze–thawing-disrupted mitochondria. As expected, ABA at concentrations from 5 to 25 lM did not cause sig- nificant changes in enzyme activity (data not shown). ABA, cATR or Oligo CCCP or Rot 10 0 R F I 1 min Mit Fig. 4. Effect of abamectin (ABA, 25 lM) on the membrane potential of glutamate plus malate-energized rat liver mitochondria. Assay conditions are described in Section 2. The figure is representative of three experiments with different mitochondrial preparations. Arrows indicate addition of compounds. Mit: mito- chondrial suspension 1 mg/ml. Oligo: oligomycin 1 lg/ml. cATR: carboxyatractylo- side 1 lM. CCCP: CCCP 1 lM. Rot: rotenone 2.5 lM. RFI: relative fluorescence intensity. 3.6. Effect of abamectin on ADP-induced depolarization of Dw The purpose of this assay was to determine whether ABA inhib- its ADP-induced depolarization of Dw by interference with ANT. Carboxyatractyloside was used as a positive control for direct ANT inhibition. ABA caused significant, concentration-dependent inhibition of ADP-stimulated depolarization of Dw (Fig. 7). 4. Discussion Mitochondrial dysfunction is a fundamental pathogenic mecha- nism that leads to several significant toxicities in mammals, espe- 0 50 ** ** ** ** AT P as (n m ol P i. m in C Olig o 5 10 15 25 0 25 50 75 100 125 * * * * ** Abamectin (μM) AT P as e ac tiv ity (n m ol P i. m in -1 . m g pr ot ei n -1 ) B Fig. 6. Effects of abamectin (ABA) on ATPase activity in intact-uncoupled mito- chondria in the presence of CCCP (A) and in freeze–thawing-disrupted rat liver mitochondria (B). Assay conditions are described in Section 2. Values represent the mean ± S.E. mean of three experiments with different mitochondrial preparations. C: control, only 0.1% DMSO. Oligo: oligomycin 1 lg/ml. ⁄,⁄⁄Significantly different from control (⁄P < 0.05 and ⁄⁄P < 0.01). C cA TR 5 10 15 25 0 25 50 75 100 ** ** ** ** ** Abamectin (μM) A D P -in du ce d de po la riz at io n of Δψ (% o f t ot al ) Fig. 7. Effect of abamectin (ABA) on ADP-induced depolarization of Dw. Assay conditions are described in Section 2. Values represent the mean ± S.E. mean of three experiments with different mitochondrial preparations. C: control, only 0.1% DMSO. cATR: carboxyatractyloside 5 lM. ⁄⁄Significantly different from control (P < 0.01). J.C. Castanha Zanoli et al. / Toxicology in Vitro 26 (2012) 51–56 55 cially those associated with the liver (Szewczyk and Wojtczak, 2002; Amacher, 2005). To assess the potential involvement of mitochondria in ABA-related hepatotoxicity, we assessed its effects on the bioenergetics of rat liver mitochondria. The results obtained using mitochondria energized with glutamate + malate (electron donors to complex I), succinate (electron donor to complex II) and TMPD/ascorbate (artificial donor of electrons to complex IV) showed that ABA inhibits state-3 respiration in a concentration- dependent manner at concentrations from 5 to 25 lM. According to Chance and Williams (1955), state-3 respiration involves mito- chondria, ADP and a respiratory substrate, and the speed of ADP phosphorylation is the limiting factor of the process. The inhibition observed in the three experiments may result from the direct ac- tion of abamectin on the respiratory chain, or from an inhibitory effect on FoF1-ATPase or ANT. It is possible to distinguish between inhibition of oxidative phosphorylation and inhibition of the electron transport chain by using an uncoupler-stimulated respiration test. If inhibition occurs in electron transport chain, uncoupler-stimulated oxygen con- sumption will be inhibited. If the tested compound instead acts on the oxidative phosphorylation, it will be innocuous. We con- ducted such a test using CCCP as an uncoupler and succinate as the substrate. Mitochondrial oxygen consumption was not inhib- ited by ABA but was inhibited for KCN (respiratory chain complex IV inhibitor), indicating that the inhibition of state-3 respiration by the compound does not occur through direct action on the respira- tory chain. The effect is probably due to interaction with FoF1-ATP- ase and/or the ADP/ATP translocator because it is similar to those of oligomycin, a specific inhibitor of FoF1-ATPase, and carboxya- tractyloside, an ANT inhibitor. In addition, mitochondrial oxygen consumption inhibited by 25 lM ABA was further stimulated with 1 lM CCCP, demonstrating that the mitochondrial respiratory chain was not inhibited (data not shown). The complex I (NADH dehydrogenase) is the most vulnerable complex of the electron transport chain. The smaller, simpler com- plex II contains succinate dehydrogenase, the only enzyme of the Krebs cycle linked to the inner mitochondrial membrane (Boelster- li, 2007). We corroborated our results cited in the item 3.5 that saw no ABA effect on NADH dehydrogenase and succinate dehydrogenase. ABA did not dissipate membrane potential, as do inhibitors of respiratory chain complexes, such as rotenone and uncoupling substances such as CCCP, i.e., those capable of acting on the linkage between ATP synthesis and electron transport. Our results support the hypothesis, proposed earlier, that ABA behaves similarly to oli- gomycin and/or carboxyatractyloside, indicating that the toxic mechanism of ABA involves direct action on FoF1-ATPase and/or ANT. Because ATP is an essential metabolic component, interference with its synthesis or use is the mechanism by which many xenobi- otics express acute or chronic toxicity (Meyer and Kulkarni, 2001). ABA significantly inhibited the synthesis of ATP at 10 lM and reached a maximum effect at 15 lM. The ANT is an important component of the mitochondrial machinery of ATP synthesis because of its intrinsic adenine nucle- otide translocase activity. ANT participates in both pathological (mitochondrial permeability transition formation/regulation and cell death) and physiological (adenine nucleotide exchange) mito- chondrial events, making it a prime target for drug-induced toxic- ity (Oliveira and Wallace, 2006). To demonstrate ABA-induced inhibition of ATPase and/or ANT, we evaluated its effects in the activity of ATPase using intact-uncoupled and freeze–thawing-dis- rupted mitochondria with an excess of ATP, a condition that drives the enzyme to operate in the reverse direction, hydrolyzing ATP (Bracht et al., 2003), and also in the ADP-induced depolarization of Dw. We saw more significant stimulation of ATPase activity in intact-uncoupled mitochondria than in disrupted mitochondria, which taken together with the observed inhibition of ADP-induced depolarization of Dw indicates that abamectin more specifically inhibits ANT than FoF1-ATPase. In conclusion, the present study shows that ABA perturbs the mitochondrial bioenergetics through different mechanisms and that its effect on the adenine nucleotide translocator (ANT) is more potent than on FoF1-ATPase. These effects constitute a potential mechanism for ABA toxicity in liver cells, which could contribute to the toxicological effects of ABA described in animals and human. 5. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). Results will be pre- sented by Juliana Carla Castanha Zanoli to the Faculdade de Medi- cina Veterinária de Araçatuba, Universidade Estadual Paulista ‘‘Júlio de Mesquita Filho’’, in partial fulfillment of the requirements for the Master degree in Ciência Animal. References Agarwal, A.K., 1998. Avermectin. In: Wexler, P. (Ed.), Encyclopedia of Toxicology. Academic Press, San Diego, pp. 89–90. Amacher, D.E., 2005. Drug-associated mitochondrial toxicity and its detection. Curr. Med. Chem. 12, 1829–1839. Boelsterli, U.A., 2007. Disruption of mitochondrial function and mitochondria- mediated toxicity. In: Mechanistic Toxicology: The Molecular Basis of How Chemicals Disrupt Biological Targets, Second ed. CRC Press, Boca Raton, pp. 357–389. Bracht, A., Ishii-Iwamoto, E.L., Salgueiro-Pagadigorria, C.L., 2003. Estudo do metabolismo energético em mitocôndrias isoladas de tecido animal. In: Bracht, A., Ishii-Iwamoto, E.L. (Eds.), Métodos de Laboratório em Bioquímica. Manole, Barueri, pp. 227–247. Cain, K., Skilleter, D.N., 1987. Preparation and use of mitochondria in toxicological research. In: Snell, K., Mullock, B. (Eds.), Biochemical Toxicology. IRL Press, Oxford, pp. 217–254. Chance, B., Williams, G.R., 1955. The respiratory chain and oxidative phosphorylation. Adv. Enzymol. 17, 65–134. Chung, K., Yang, C.C., Wu, M.L., Deng, J.F., Tsai, W.J., 1999. Agricultural avermectins: an uncommon but potentially fatal cause of pesticide poisoning. Ann. Emerg. Med. 34, 51–57. Eissa, F.I., Zidan, N.A., 2010. Haematological, biochemical and histopathological alterations induced by abamectin and Bacillus thuringiensis in male albino rats. Acta Biol. Hung. 61, 33–44. El-Shenawy, N.S., 2010. Effects of insecticides fenitrothion, endosulfan and abamectin on antioxidant parameters of isolated rat hepatocytes. Toxicol. in Vitro 24, 1148–1157. 56 J.C. Castanha Zanoli et al. / Toxicology in Vitro 26 (2012) 51–56 Fiske, C.H., Subbarow, Y., 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66, 375–400. Garcia, A.F., Medeiros, H.C.D., Maioli, M.A., Lima, M.C., Rocha, B.A., Costa, F.B., Curti, C., Groppo, M., Mingatto, F.A., 2010. Comparative effects of lantadene A and its reduced metabolite on mitochondrial bioenergetics. Toxicon 55, 1331– 1337. Guillouzo, A., 1998. Liver cell models in in vitro toxicology. Environ. Health Perspect. 106, 511–532. Hayes, W.J., Laws, E.R., 1990. Handbook of Pesticide Toxicology Classes of Pesticides, vol. 2. Academic Press Inc., New York, p. 3. Hsu, D.Z., Hsu, C.H., Huang, B.M., Liu, M.Y., 2001. Abamectin effects on aspartate aminotransferase and nitric oxide in rats. Toxicology 165, 189–193. Kolar, L., Erzen, N.K., Hogerwerf, L., Van Gestel, C.A.M., 2008. Toxicity of abamectin and doramectin to soil invertebrates. Environ. Pollut. 151, 182–189. Lankas, G.R., Gordon, L.R., 1989. Toxicology. In: Campbell, W.C. (Ed.), Ivermectin and Abamectin. Springer-Verlag, New York, pp. 10–142. Lemasters, J.J., Hackenbrock, C.R., 1976. Continuous measurement and rapid kinetics of ATP synthesis in rat liver mitochondria, mitoplasts and inner membrane vesicles determined by firefly-luciferase luminescence. Eur. J. Biochem. 67, 1–10. Meyer, S.A., Kulkarni, A.P., 2001. Hepatotoxicity. In: Hodgson, E., Smart, R.C. (Eds.), Introduction to Biochemical Toxicology, third ed. John Wiley & Sons, New York, pp. 487–507. Mingatto, F.E., Santos, A.C., Rodrigues, T., Pigoso, A.A., Uyemura, S.A., Curti, C., 2000. Effects of nimesulide and its reduced metabolite on mitochondria. Br. J. Pharmacol. 131, 1154–1160. Mingatto, F.E., Dorta, D.J., Santos, A.B., Carvalho, I., Silva, C.H.T.P., Silva, V.B., Uyemura, S.A., Santos, A.C., Curti, C., 2007. Dehydromonocrotaline inhibits mitochondrial complex I. A potential mechanism accounting for hepatotoxicity of monocrotaline. Toxicon 50, 724–730. Mitchell, P., 1961. Coupling of phosphorylation to electron and hydrogen transfer by a chemiosmotic type of mechanism. Nature 191, 144–148. O’Brien, T.M., Oliveira, P.J., Wallace, K.B., 2008. Inhibition of the adenine nucleotide translocator by N-acetyl perfluorooctane sulfonamides in vitro. Toxicol. Appl. Pharmacol. 227, 184–195. Oliveira, P.J., Wallace, K.B., 2006. Depletion of adenine nucleotide translocator protein in heart mitochondria from doxorubicin-treated rats. Relevance for mitochondrial dysfunction. Toxicology 220, 160–168. Pedersen, P.L., Greenawalt, J.W., Reynafarje, B., Hullihen, J., Decker, G.L., Soper, J.W., Bustamente, E., 1978. Preparation and caracterization of mitochondria and submitochondrial particles of rat liver and liver-derived tissues. Methods Cell. Biol. 20, 411–481. Seixas, J.N., Peixoto, P.V., Armién, A.G., Jabour, F.F., Brito, M.F., 2006. Clinical and pathogenetic aspects of abamectin poisoning in calves. Pesq. Vet. Bras. 26, 161– 166. Szewczyk, A., Wojtczak, L., 2002. Mitochondria as a pharmacological target. Pharmacol. Rev. 54, 101–127. Yang, C.C., 2008. Avermectin Poisoning, in: Abstracts of the XXVIII International Congress of the European Association of Poison Centres and Clinical Toxicologists, May 6–9, Seville, Spain. Clin. Toxicol. 46, 351-421. Zanotti, A., Azzone, G.F., 1980. Safranine as membrane potential probe in rat liver mitochondria. Arch. Biochem. Biophys. 201, 255–265. Abamectin affects the bioenergetics of liver mitochondria: A potential mechanism of hepatotoxicity 1 Introduction 2 Materials and methods 2.1 Chemicals 2.2 Animals 2.3 Isolation of intact and disrupted rat liver mitochondria 2.4 Mitochondrial respiration assay 2.5 Estimation of mitochondrial membrane potenti 2.6 ATP quantification 2.7 Mitochondrial ATPase activity 2.8 Determination of enzyme activity related to mitochondrial respiratory chain (NADH and succinate dehydrogenase) 2.9 Inhibition of ADP-induced depolarization of 2.10 Statistical analysis 3 Results 3.1 Effects of abamectin on mitochondrial respiration 3.2 Effect of abamectin on mitochondrial membran 3.3 Effect of abamectin on mitochondrial ATP levels 3.4 Effects of abamectin on the FoF1-ATPase activity 3.5 Effect of abamectin on NADH and succinate dehydrogenase activities 3.6 Effect of abamectin on ADP-induced depolariz 4 Discussion 5 Conflict of interest statement Acknowledgements References