Toxicology in Vitro 27 (2013) 570–579 Contents lists available at SciVerse ScienceDirect Toxicology in Vitro journal homepage: www.elsevier .com/locate / toxinvi t The role of mitochondria and biotransformation in abamectin-induced cytotoxicity in isolated rat hepatocytes Marcos A. Maioli a, Hyllana C.D. de Medeiros a, Marieli Guelfi a, Vitor Trinca b, Flávia T.V. Pereira b, Fábio E. Mingatto a,⇑ a Laboratório de Bioquímica Metabólica e Toxicológica (LaBMeT), UNESP – Univ Estadual Paulista, Campus de Dracena, 17900-000 Dracena, SP, Brazil b Laboratório de Morfofisiologia da Placenta e Embrião (L@MPE), 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 17 July 2012 Accepted 29 October 2012 Available online 6 November 2012 Keywords: Abamectin Hepatotoxicity Calcium ATP Necrosis 0887-2333 � 2012 Elsevier Ltd. http://dx.doi.org/10.1016/j.tiv.2012.10.017 ⇑ Corresponding author. Tel.: +55 18 3821 8158; fa E-mail address: fmingatto@dracena.unesp.br (F.E. Open access under the Elsevi a b s t r a c t Abamectin (ABA), which belongs to the family of avermectins, is used as a parasiticide; however, ABA poi- soning can impair liver function. In a previous study using isolated rat liver mitochondria, we observed that ABA inhibited the activity of adenine nucleotide translocator and FoF1-ATPase. The aim of this study was to characterize the mechanism of ABA toxicity in isolated rat hepatocytes and to evaluate whether this effect is dependent on its metabolism. The toxicity of ABA was assessed by monitoring oxygen con- sumption and mitochondrial membrane potential, intracellular ATP concentration, cell viability, intracel- lular Ca2+ homeostasis, release of cytochrome c, caspase 3 activity and necrotic cell death. ABA reduces cellular respiration in cells energized with glutamate and malate or succinate. The hepatocytes that were previously incubated with proadifen, a cytochrome P450 inhibitor, are more sensitive to the compound as observed by a rapid decrease in the mitochondrial membrane potential accompanied by reductions in ATP concentration and cell viability and a disruption of intracellular Ca2+ homeostasis followed by necro- sis. Our results indicate that ABA biotransformation reduces its toxicity, and its toxic action is related to the inhibition of mitochondrial activity, which leads to decreased synthesis of ATP followed by cell death. �2012 Elsevier Ltd. Open access under the Elsevier OA license. 1. Introduction Avermectins are metabolites derived from the fermentation of the fungi Streptomyces avermitilis; these metabolites belong to the family of macrocyclic lactones and exhibit extraordinarily po- tent anthelmintic activity (Burg et al., 1979; Fisher and Mrozik, 1989). Abamectin (ABA) is a mixture of avermectins containing P80% B1a and 620% B1b (Meister, 1992; Zeng et al., 1996; Agarwal, 1998). Avermectin B1a and B1b differ chemically by the presence of a methylene or ethylene group at C-26 (Zeng et al., 1996). Accord- ing to Hayes and Laws (1990), these molecules have similar biolog- ical activities and toxicological properties. ABA is widely used because of its potent anthelmintic and insecticidal action and wide spectrum of action. ABA is also used as an insecticide to control cit- rus, nut culture and household pests, such as fire ants (Elbetieha and Daas, 2003). In veterinary medicine, ABA is administered to animals in a systematic way to control endoparasites and ectopar- asites (Shoop et al., 1995). The mechanism of ABA action is related to its effect on the c-aminobutyric acid (GABA) system and Cl� channels. GABA recep- tors are responsible for regulating the neural basal tone of the x: +55 18 3821 8208. Mingatto). er OA license. brain (Turner and Schaeffer, 1989) and are in virtually all neurons of the central nervous system (CNS). The symptoms of ABA poison- ing exhibited in laboratory animals include pupil dilation, vomit- ing, convulsions and/or tremors and coma (Lankas and Gordon, 1989). In addition, some studies have reported genotoxic effects of ABA (Molinari et al., 2010). As demonstrated by the in vivo studies (Lowenstein et al., 1996; Hsu et al., 2001) and the in vitro study conducted with isolated hepatocytes (El-Shenawy, 2010), the liver can also be affected by ABA. ABA caused an increase in the concentration of the enzyme aspartate aminotransferase (AST) in serum in vivo and an increase in the concentration of AST and alanine aminotransferase (ALT) in vitro, which are used as indicators of damage to the hepatic parenchymal cells (Klaassen and Eaton, 1991). We previously dem- onstrated that ABA inhibits the activity of FoF1-ATPase and adenine nucleotide translocator (ANT) when added at micromolar concen- trations to isolated rat liver mitochondria, an effect associated with significantly reduced ATP synthesis (Castanha Zanoli et al., 2012). FoF1-ATPase is an enzyme present in the inner mitochondrial membrane that is responsible by ATP synthesis driven by the pro- ton electrochemical gradient generated in the respiratory chain. The main components of the enzyme are Fo, an integral membrane protein that works as a proton channel, and F1, a hydrophilic moiety which contains the catalytic and regulatory sites (Hatefi, http://dx.doi.org/10.1016/j.tiv.2012.10.017 mailto:fmingatto@dracena.unesp.br http://dx.doi.org/10.1016/j.tiv.2012.10.017 http://www.sciencedirect.com/science/journal/08872333 http://www.elsevier.com/locate/toxinvit http://www.elsevier.com/open-access/userlicense/1.0/ http://www.elsevier.com/open-access/userlicense/1.0/ M.A. Maioli et al. / Toxicology in Vitro 27 (2013) 570–579 571 1993; Pedersen, 1996). ANT is other important component of the mitochondrial machinery of ATP synthesis because of its intrinsic adenine nucleotide translocase activity. ANT has been involved in both pathological (mitochondrial permeability transition forma- tion/regulation and cell death) and physiological (adenine nucleo- tide exchange) mitochondrial events, making it a prime target for drug-induced toxicity (Oliveira and Wallace, 2006). The xenobiotic metabolism in the liver is accomplished by cyto- chrome P450 and its main function is to increase the polarity of these substances, so excretion occurs more easily (Oga, 2008). However, this process is responsible for the toxic effects of numer- ous chemical compounds. The metabolites may cause adverse ef- fects in the animal (Ioannides and Lewis, 2004; Mingatto et al., 2007; Maioli et al., 2011) by changing a fundamental cellular com- ponent (mitochondria, for example) at the cellular and molecular level, thus modulating its function (Meyer and Kulkarni, 2001). Due to the important functions of the liver in animals and pre- vious studies that indicated the occurrence of liver damage after the use of ABA, this study aims to characterize the mechanisms of ABA toxicity on parameters related to bioenergetics and cell death and determine whether the toxicity induced by the com- pound is due to a possible activation following its metabolism in the liver. 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, SP, Brazil), proadifen was purchased from Sigma– Aldrich (St. Louis, MO, USA), and sodium pentobarbital was a gift from Cristália (Itapira, SP, Brazil). All other reagents were of the highest commercially available grade. Abamectin and proadifen were dissolved in anhydrous dimethyl sulfoxide (DMSO). All stock solutions were prepared using glass-distilled deionized water. 2.2. Animals Male Wistar rats aged 7–8 weeks and weighing approximately 200 g, were used in this study. The animals, which were obtained from the Central Bioterium of UNESP – Univ Estadual Paulista, Campus de Botucatu, SP, Brazil, were maintained with a maximum of 4 rats per cage under standard laboratory conditions with water and food provided ad libitum. The experimental protocols were ap- proved by the Ethical Committee for the Use of Laboratory Animals of the UNESP – Univ Estadual Paulista, Campus de Dracena, SP, Brazil. 2.3. Isolation and incubation of hepatocytes For the surgical procedure, the rats were anesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight). The hepatocytes were isolated by a collagenase perfusion of the liver as described previously (Guguen-Guillouzo, 1992). The hepatocyte viability after isolation was determined by Trypan blue (0.16%) uptake, and the initial cell viability in all experiments was more than 85%. The hepatocytes were suspended in Krebs-Hense- leit buffer, pH 7.4, containing 12.5 mM Hepes and 0.1% bovine ser- um albumin (BSA), and maintained at 4 �C. The cells (1 � 106/mL) were incubated in 25-mL Erlenmeyer flasks, which were main- tained under constant agitation (30 rpm) at 37 �C under a 95% O2 and 5% CO2 atmosphere. The reactions in the experiments of cell viability, cellular ATP content, mitochondrial membrane potential, release of cytochrome c, caspase 3 activity and necrotic cell death were initiated by the addition of abamectin (ABA) at concentra- tions of 25, 50, 75 and 100 lM. Aliquots (1 mL) of the suspension were removed from the mixture at appropriate times for the deter- mination of cell death and biochemical parameters. In some exper- iments, the cells were incubated with 100 lM proadifen 15 min before the addition of ABA. 2.4. Oxygen uptake Oxygen uptake by the isolated hepatocytes was monitored using a Clark-type oxygen electrode (Strathkelvin Instruments Limited, Glasgow, Scotland, UK). The respiration buffer contained 250 mM sucrose, 2 mM KH2PO4, 10 mM HEPES, pH 7.2, 0.5 mM EGTA, 0.5% BSA, and 5 mM MgCl2, at 37 �C. The cells were treated with 0.002% digitonin, and state 4 and state 3 mitochondrial respi- ration rates were measured in the presence of 1 lg/mL oligomycin and 2 mM ADP, respectively (Moreadith and Fisckum, 1984). ABA at concentrations of 5, 10, 15 and 25 lM was added to the medium immediately after the initiation of state 3 or state 4 respirations. 2.5. Mitochondrial membrane potential The mitochondrial membrane potential was determined using the fluorescent probe TMRM (tetramethyrodamine, methyl ester). The cell suspensions incubated with different concentrations of abamectin were collected and centrifuged at 50g for 5 min. The pellet was suspended and incubated for 10 min at 37 �C with TMRM solution at a final concentration of 6.6 lM. After the incuba- tion, the samples were centrifuged twice at 50g for 5 min, and the pellet was suspended with 1 ml of Triton X-100, 0.1% (v/v). Subse- quently, the samples were centrifuged at 2000g for 5 min, and the fluorescence of the TMRM captured and retained by the mitochon- dria was determined in the supernatant using a fluorescence spec- trophotometer RF-5301 PC (Shimadzu, Tokyo, Japan) at excitation and emission wavelengths of 485 and 590 nm, respectively. The re- sults are expressed as a percentage of the fluorescence intensity over the control group. 2.6. Cellular ATP content Cellular ATP content was determined by the firefly luciferin– luciferase assay. The cell suspension was centrifuged at 50g for 5 min at 4 �C, and the pellet containing the hepatocytes was trea- ted with 1 mL of ice-cold 1 M HClO4. After centrifugation at 2000g for 10 min at 4 �C, aliquots (100 lL) of the supernatant were neutralized with 65 lL of 2 M KOH, suspended in 100 mM Tris–HCl, pH 7.8 (1 mL final volume), and centrifuged again. Biolu- minescence was measured in the supernatant with a Sigma– Aldrich assay kit according to the manufacturer’s instructions using a SIRIUS Luminometer (Berthold, Pforzheim, Germany). 2.7. Evaluation of cell viability Cell viability was assessed by the leakage of alanine transami- nase (ALT) and aspartate transaminase (AST) from hepatocytes. After incubation with ABA at concentrations of 25, 50, 75 and 100 lM the cell suspensions were collected at time 0, 30, 60, 90 and 120 min and centrifuged (50g for 5 min). The presence of ALT and AST in the supernatant was determined using Enzyme Activity Assay Kits (Bioclin, Quibasa, Brazil) according to the man- ufacturer’s instructions. The absorbance was measured at 340 nm with a spectrophotometer DU-800 (Beckman Coulter, Fullerton, CA, USA). Enzyme activity in the supernatant is expressed as a per- centage of the total activity, which was determined by lysing the cells with 0.5% Triton X-100. 0.0 12.5 25.0 37.5 50.0 ** ** ** ** 0 5 10 15 20 25 0 15 30 45 60 ** ** ** * Abamectin ( μM) O xy ge n C on su m pt io n (n m ol O 2/ m in . 10 6 c el ls ) A B Fig. 1. Effects of abamectin (ABA) on glutamate-plus-malate-supported (A) or succinate-supported (B) state 3 (ADP stimulated) respiration of mitochondria in digitonin-permeabilized isolated rat hepatocytes. The figure is representative of five experiments with different cell preparations. ⁄,⁄⁄Significantly different from the control (without ABA) (P < 0.05 and P < 0.01, respectively). 572 M.A. Maioli et al. / Toxicology in Vitro 27 (2013) 570–579 2.8. Intracellular Ca2+ homeostasis Hepatocytes (2 � 106/ml) were incubated in Krebs-Henseleit medium supplemented with 2% BSA, 12.5 mM HEPES and 10 mM glucose, pH 7.4. In this medium, 0.005% pluronic acid and 5 lM Fura-2 acetoxymethyl ester (Fura-2 AM) were added. The 0 30 60 90 120 0 25 50 75 100 ABA 25 μM ABA 25 μM + P 0 30 60 90 120 0 25 50 75 100 ABA 75 μM ABA 75 μM + P ** ## ## ## ## ## ## ## ## ΔΨ m (% o f t ot al ) Ti Fig. 2. Effects of abamectin (ABA) on the mitochondrial membrane potential assessed by isolated rat hepatocytes (106 cells/ml) in the absence or presence of 100 lM proadifen preparations. ##Significantly different from control (without ABA) at the correspon corresponding time (P < 0.05 and P < 0.01, respectively). hepatocytes were maintained under constant agitation at 32 �C for 60 min to capture the probe. The cell suspension loaded with Fura-2 AM was collected and subjected to two centrifugations at 50g for 3 min to remove resid- ual Fura-2 AM and maintained at 4 �C for later use. The fluores- cence of Ca2+ was determined by the ratio of the excitation wavelengths at 340 and 380 nm and emission wavelength at 505 nm using the fluorescence spectrophotometer RF-5301 PC (Shimadzu, Tokyo, Japan). The calibration and calculations in [Ca2+]c were performed as previously described (Grynkiewicz et al., 1985). Maximum fluorescence (Fmax) was obtained by the addition of 1% Triton X-100, and minimum fluorescence (Fmin) was obtained by the addition of 10 mM EGTA. The equilibrium con- stant for the calculations was 225 nM. Changes in free [Ca2+]c in the cytoplasm of hepatocytes were evaluated with increasing addi- tions of ABA (25, 50, 75 and 100 lM) every 300 s. 2.9. Release of cytochrome c The release of cytochrome c was determined as previously de- scribed (Appaix et al., 2000). The hepatocytes (2.7 mg protein/ml) were incubated in Krebs-Henseleit medium supplemented with BSA (2 mg/mL), 0.002% digitonin and different concentrations of abamectin at 25 �C for 30 min. After the incubation, the cells were centrifuged at 10,000g for 30 min at 4 �C, and the supernatant was collected and filtered through a 0.2 lm Millipore membrane. The absorbance was determined in a spectrophotometer DU-800 (Beckman Coulter, Fullerton, CA, USA) by the difference in absor- bance at wavelengths 414 and 600 nm. The results are expressed in nmol cytochrome c released/106 cells using a molar extinction coefficient (e) of 100 mM�1 cm�1. 2.10. Caspase 3 activity The assessment of caspase 3 activity was performed using a Caspase 3 assay kit (Sigma–Aldrich). The hepatocytes were 0 30 60 90 120 0 25 50 75 100 ABA 50 μM ABA 50 μM + P ## ## ## ** * 0 30 60 90 120 0 25 50 75 100 ABA 100 μM ABA 100 μM + P ** ## ## ## ## ## ## ## ##** * me (min) the uptake of the fluorescent probe tetramethylrhodamine methyl ester (TMRM) in (P). The results represent the mean ± SEM of six experiments with different cell ding time (P < 0.01). ⁄,⁄⁄Significantly different from ‘‘without proadifen’’ at the 0 30 60 90 120 0 5 10 15 20 25 Control Control + P 0 30 60 90 120 0 5 10 15 20 25 ABA 25 μM ABA 25 μM + P * ** ** # # # 0 30 60 90 120 0 5 10 15 20 25 ABA 50 μM ABA 50 μM + P * ## ## ## ## ## ## ## 0 30 60 90 120 0 5 10 15 20 25 ABA 75 μM * ## ## ## ** ## ** ## ABA 75 μM + P ** ## 0 30 60 90 120 0 5 10 15 20 25 ABA 100 μM ## ** # # ** ## ## ## ABA 100 μM + P ## ## **** Time (min) A TP (n m ol /1 06 c el ls ) Fig. 3. Effects of abamectin (ABA) on the intracellular concentration of ATP in isolated rat hepatocytes (106 cells/ml) in the absence or presence of 100 lM proadifen (P). The results represent the mean ± SEM of five different cell preparations. #,##Significantly different from control (without ABA) at the corresponding time (P < 0.05 and P < 0.01, respectively). ⁄,⁄⁄Significantly different from ‘‘without proadifen’’ at the corresponding time (P < 0.05 and P < 0.01, respectively). M.A. Maioli et al. / Toxicology in Vitro 27 (2013) 570–579 573 collected and centrifuged at 600g for 5 min and suspended in 1 mL of phosphate buffered saline (PBS). Further centrifugation was per- formed, and the precipitate was incubated for 15 min at 4 �C with 200 lL of lysis buffer for the release of caspase 3, and 300 lL of PBS was then added. The lysed cell suspension was centrifuged at 14,000g for 15 min at 4 �C, and the supernatant was collected. Ali- quots of 50 lL of supernatant were used to assess the activity of caspase 3 according to the manufacturer’s instructions. Fluores- cence was determined using the fluorescence spectrophotometer RF-5301 PC (Shimadzu, Tokyo, Japan) at wavelengths of 360 and 460 nm for excitation and emission, respectively. The results are expressed as pmol of AMC/min/mL. 2.11. Necrotic cell death Samples of cells (200 lL) were collected and centrifuged at 50g for 5 min, and the precipitate was suspended in Krebs/Henseleit medium, pH 7.4, and incubated with Hoechst 33342 (8 lg/mL) and Propidium Iodide (5 lM) dyes for 15 min at room temperature in the dark. After incubation, the samples were centrifuged twice at 50g for 5 min to remove excess dye. After the washes, the hepato- cytes were suspended in 50 lL of Krebs/Henseleit medium, pH 7.4. The cells were analyzed with a fluorescence microscope (DM 2500 type, Leica, Rueil-Malmaison, France), and the percentage of necro- tic cells was quantitated using the Qwin 3.0 software. 2.12. Statistical analysis Data are expressed as the mean ± standard error of the mean (S.E.M.). The statistical significance of the differences between con- trol and the experimental groups was evaluated using one-way analysis of variance (ANOVA) followed by Dunnett’s test, and dif- ferences between the experimental groups at the same time points was evaluated using unpaired t test with Welch́s correction. Values of P < 0.05 were considered to be significant. All statistical analyses were performed using GraphPad Prism software, version 4.0 for Windows (GraphPad Software, San Diego, CA, USA). 3. Results 3.1. Effects of ABA on the respiration of mitochondria in isolated rat hepatocytes Fig. 1 shows the inhibitory effect of ABA on the glutamate- plus-malate-supported and succinate-supported state 3 (ADP- stimulated) respiration of mitochondria in digitonin-permeabilized hepatocytes. ABA has an inhibitory action on cellular respiration un- der conditions with the substrates of the respiratory chain complex I (glutamate + malate) and with the substrate of complex II (succi- nate) at the same concentrations and in a concentration-dependent manner beginning at 5 lM. ABA did not stimulate state 4 (basal) 0 30 60 90 120 0 25 50 75 100 Control Control + P 0 30 60 90 120 0 25 50 75 100 ABA 25 μM ABA 25 μM + P * ** ** ** 0 30 60 90 120 0 25 50 75 100 ABA 50 μM ABA 50 μM + P * ** ** ** # ## # # 0 30 60 90 120 0 25 50 75 100 ABA 75 μM ABA 75 μM + P ** ## ## ##**## **## 0 30 60 90 120 0 25 50 75 100 ABA 100 μM ABA 100 μM + P **##**## **## **# #### Time (min) A LT le ak ag e (% o f t ot al ) Fig. 4. Effects of abamectin (ABA) on cell viability assessed by the release of the enzyme alanine transaminase (ALT) in isolated rat hepatocytes (106 cells/ml) in the absence or presence of 100 lM proadifen (P). The results represent the mean ± SEM of six experiments with different cell preparations. #,##Significantly different from control (without ABA) at the corresponding time (P < 0.05 and P < 0.01, respectively). ⁄,⁄⁄Significantly different from ‘‘without proadifen’’ at the corresponding time (P < 0.05 and P < 0.01, respectively). 574 M.A. Maioli et al. / Toxicology in Vitro 27 (2013) 570–579 respiration (results not shown). These results indicate that ABA inhibits the oxidative phosphorylation of mitochondria as assessed in isolated hepatocytes, and the results are in agreement with those previously described that show ABA as an inhibitor of the adenine nucleotide translocator (ANT) and FoF1-ATPase in isolated mito- chondria (Castanha Zanoli et al., 2012). Proadifen (100 lM) did not present any effect on the mitochondrial respiration of hepato- cytes (results not shown). 3.2. Effects of ABA on the mitochondrial membrane potential and ATP levels in isolated rat hepatocytes The effects of ABA on the mitochondrial membrane potential and ATP levels were evaluated in the presence or absence of pro- adifen, a cytochrome P450 inhibitor (Figs. 2 and 3, respectively). The addition of increasing concentrations of ABA to the hepato- cytes (25–100 lM) resulted in a decrease in the mitochondrial membrane potential and ATP levels in a concentration- and time- dependent manner. Proadifen stimulated an ABA-induced decrease in the mitochondrial membrane potential and ATP levels (Figs. 2 and 3, respectively), suggesting that the parent drug by itself is the main factor responsible for the toxic effect on isolated hepatocytes. 3.3. Effects of ABA on cell viability in isolated rat hepatocytes The activity of ALT (Fig. 4) and AST (Fig. 5) was used to monitor the viability of hepatocytes following exposure to different concen- trations of ABA (25–100 lM) in the absence and presence of proadifen. The addition of increasing concentrations of ABA to hepatocytes resulted in decreased cell viability, as assessed by ALT and AST leakage into the incubation medium, in a concentration- and time-dependent manner (Figs. 4 and 5, respectively). A significant increase in the concentration of ALT and AST was observed with 50 lM ABA at 90 min. Proadifen stimulated the ABA-induced decrease in cell viability because the cells showed a significant release of both enzymes in the presence of ABA (Figs. 4 and 5). 3.4. Effects of ABA on intracellular Ca2+ homeostasis in isolated rat hepatocytes Intracellular Ca2+ homeostasis was evaluated by changes in the fluorescence probe Fura-2 in hepatocytes exposed to increasing concentrations of ABA (25–100 lM) in the absence of proadifen (Fig. 6). 0 30 60 90 120 0 25 50 75 100 Control Control + P 0 30 60 90 120 0 25 50 75 100 ABA 25 μM ABA 25 μM + P * * ** ** 0 30 60 90 120 0 25 50 75 100 ABA 50 μM ABA 50 μM + P ** ** # # ## # * * 0 30 60 90 120 0 25 50 75 100 ABA 75 μM ** ## ## ## ** ABA 75 μM + P ## ** 0 30 60 90 120 0 25 50 75 100 ABA 100 μM ** ## ** ## ** ## ** # ## ## ABA 100 μM + P Time (min) A ST le ak ag e (% o f t ot al ) Fig. 5. Effects of abamectin (ABA) on cell viability assessed by the release of the enzyme aspartate transaminase (AST) in isolated rat hepatocytes (106 cells/ml) in the absence or presence of 100 lM proadifen (P). The results represent the mean ± SEM of six experiments with different cell preparations. #,##Significantly different from control (without ABA) at the corresponding time (P < 0.05 and P < 0.01, respectively). ⁄,⁄⁄Significantly different from ‘‘without proadifen’’ at the corresponding time (P < 0.05 and P < 0.01, respectively). 0 20 0 40 0 60 0 80 0 10 00 12 00 14 00 16 00 18 00 0 100 200 300 400 500 600 ABA 25μM 25μM 25μM 25μM Time (sec) [C a2+ ] c (n M ) 50 μM 75 μM 100 μM25 μM Fig. 6. Effects of abamectin (ABA) on intracellular Ca2+ homeostasis in isolated rat hepatocytes (2 � 106 cells/ml) permeabilized with pluronic acid (0.005%). The results represent the mean ± SEM of five experiments with different cell preparations. M.A. Maioli et al. / Toxicology in Vitro 27 (2013) 570–579 575 The cytosolic Ca2+ concentration was increased after the addi- tion of 25 lM ABA and did not change following the addition of higher concentrations (50, 75 and 100 lM) of the drug. 3.5. Effects of ABA on cytochrome c release in isolated rat hepatocytes The release of cytochrome c by the mitochondria was deter- mined in hepatocytes exposed to increasing concentrations of ABA (25–100 lM) in the absence of proadifen. The addition of ABA to the incubation medium of hepatocytes did not result in a significant release of mitochondrial cytochrome c (results not shown). 3.6. Effects of ABA on caspase 3 activity in isolated rat hepatocytes Caspase 3 activity was evaluated in hepatocytes previously incubated with proadifen and exposed to increasing concentra- tions of ABA (25–100 lM). However, the addition of ABA to the incubation medium did not cause caspase 3 activation in hepato- cytes throughout the experimental period (results not shown). 3.7. Effects of ABA on the induction of necrotic cell death in isolated rat hepatocytes After 120 min of incubation, cell necrosis was evaluated by Hoechst-propidium-iodide double staining in hepatocytes in the absence or previously incubated with proadifen and exposed to increasing concentrations of ABA (25–100 lM) (Fig. 7). 576 M.A. Maioli et al. / Toxicology in Vitro 27 (2013) 570–579 ABA triggers cell death by necrosis in a concentration- and time-dependent manner, becoming significant at 60 min for con- centrations of 75 and 100 lM (Bottom panel). Fifty micromolar of ABA triggered necrosis after only 120 min of incubation. Proadi- fen stimulated the ABA-induced cell necrosis. 4. Discussion In this study, we used isolated rat hepatocytes to study the tox- icity mechanism induced by ABA in vitro and the influence of bio- transformation of the drug. The interference of ABA in the functioning of the mitochondrial respiratory chain in isolated rat Fig. 7. (Top panel) Representative figures showing the effects of abamectin (ABA) on cel and propidium iodide. (A, B, C, D and E) 0, 25, 50, 75 and 100 lM ABA, respectively, at 0 m 50, 75 and 100 lM ABA, respectively, at 120 min, without proadifen, (K, L, M, N and O) 0, Quantitation of necrotic cells expressed as the percentage of total cells counted. The preparations in the absence or presence of 100 lM proadifen. #,##Significantly differe respectively). ⁄⁄Significantly different from ‘‘without proadifen’’ at the corresponding tim hepatocytes was monitored by measuring oxygen consumption. The results showed a clear inhibition of the rate of oxygen con- sumption in state 3 of mitochondrial respiration with both sub- strates of complex I (glutamate + malate) and complex II (succinate) at all of the tested concentrations (5–25 lM). These re- sults are consistent with those obtained by Castanha Zanoli et al. (2012), in which the effects of ABA on the isolated mitochondria of rat liver were evaluated and an inhibitory effect on the ANT and FoF1-ATPsintase was shown. During the biotransformation of xenobiotics in the liver, the metabolites generated can be even more toxic than the parent compound (Ioannides and Lewis, 2004). In a study using rat liver l death by necrosis at 0 and 120 min as monitored by the fluorescent dyes, Hoechst in, representatives of experiments without or with proadifen, (F, G, H, I and J) 0, 25, 25, 50, 75 and 100 lM ABA, respectively, at 120 min, with proadifen. (Bottom panel) results are shown as the mean ± S.E.M. of three experiments with different cell nt from control (without ABA) at the corresponding time (P < 0.05 and P < 0.01, e (P < 0.01). 0 30 60 90 120 0 25 50 75 100 Control Control + P 0 30 60 90 120 0 25 50 75 100 ABA 25 μM ABA 25 μM + P 0 30 60 90 120 0 25 50 75 100 ABA 50 μM ABA 50 μM + P ## # 0 30 60 90 120 0 25 50 75 100 ABA 75 μM ABA 75 μM + P ## ## ## ## ## ** ** 0 30 60 90 120 0 25 50 75 100 ABA 100 μM ABA 100 μM + P ## #### ## ## ** ** ** N ec ro tic c el ls (% ) Time (min) Fig. 7. (continued) M.A. Maioli et al. / Toxicology in Vitro 27 (2013) 570–579 577 microsomes, Zeng et al. (1996) showed that the major metabolites produced from abamectin are 300-O-Desmethyl B1a (300-ODMe B1a), 24-Hydroxymethyl B1a (24 OHMe-B1a) and 26-Hydroxymethyl B1a (26 OHMe-B1a). The authors attributed the metabolism of ABA to cytochrome P450 isoforms 1A1 and 3A as responsible for the metabolism of ABA, being the production of the metabolite 300- ODMe B1a attributed to isoform 3A and the production of metabo- lites 24 OHMe-B1a and 26-OHMe B1a to isoform 1A1. Therefore, to evaluate the effect of the biotransformation on ABA toxicity, the hepatocytes were incubated in the absence or presence of proadifen, a broad inhibitor of cytochrome P450 iso- forms (Khan et al., 1993; Bort et al., 1998; Mingatto et al., 2002; Somchit et al., 2009; Shi et al., 2011), which was previously shown to inhibit about 90% of the metabolism of ABA (Zeng et al., 1996). ABA metabolism interferes with the mitochondrial membrane potential because a more significant decrease in this parameter was observed in hepatocytes in the presence of proadifen. Due to the inhibition of oxidative phosphorylation and the formation of a mitochondrial membrane potential induced by ABA, a reduction in the intracellular ATP concentration is expected. This effect was observed in liver cells incubated with or without proadifen. The ef- fect was more pronounced in the cells incubated with the P450 inhibitor, indicating that the parent drug is more toxic than the metabolites. Castanha Zanoli et al. (2012) observed an inhibitory effect of ABA on the activity of ANT and FoF1-ATPase, thus blocking oxidative phosphorylation. However, the researchers did not observe a de- crease in the mitochondrial membrane potential as was observed in this study. A possible explanation for the dissipation in the mem- brane potential caused by ABA in isolated hepatocytes may be re- lated to a loss of intracellular Ca2+ homeostasis (Skulachev, 1999). When hepatocytes were exposed to 25 lM ABA, a loss of intracellu- lar ion homeostasis occurred. As the Ca2+ concentration increased in the cell cytoplasm, the mitochondria captured the surplus using the uniporter (UP) channel. According to Brookes et al. (2004), the UP ion uptake is dependent on the membrane potential, so the movement of charges due to the uptake of calcium consumes the membrane potential that was formed. Furthermore, ABA-induced mitochondrial dysfunction reduces cellular ATP levels and can promote in other organelles such as endoplasmic reticulum, the inactivation of the pump respon- sible for the maintenance of the Ca2+ ion gradient in the cyto- plasm. Invariably, the result of inhibition of the transport system is the disruption of intracellular calcium homeostasis. The increase in intracellular Ca2+ can activate proteases, phospholipases and ion-dependent endonucleases (Trump and Berzesky, 1992). The activation of proteases and phospholipases induces changes in the cytoskeleton and plasma membrane. When combined, these processes culminate in the disruption of cytoskeleton-plasma membrane interactions, which results in destabilization of the lipid bilayer, bleb formation on the cell surface and, in more severe cases, leakage and cellular necrosis (Nicotera et al., 1986; Gores et al., 1990; Sakaida et al., 1992). Fig. 8. Schematic representation of the mechanisms of ABA-induced impairment of mitochondrial bioenergetics, disruption of calcium homeostasis and necrosis in isolated rat hepatocytes. 578 M.A. Maioli et al. / Toxicology in Vitro 27 (2013) 570–579 The enzymes ALT and AST are used as indicators of damage to hepatic parenchymal cells (Klaassen and Eaton, 1991; Kaplowitz, 2001). According to Grisham (1979), an efflux of these enzymes in the liquid incubation of cells in culture indicates that there was a loss of membrane integrity. However, this efflux is not only associated with cell death and lysis but also with modifications that can be reversible (Grisham and Smith, 1984). ABA increased the concentration of ALT and AST in the liquid incubation of hepatocytes, and this effect was also influenced by pre-incubation of the cells with proadifen. The changes observed in the release of these enzymes may be a reflection of the influence of ABA on mitochondrial activity. A decrease in the efficiency of en- ergy production by the organelle affects cellular functions that are dependent on energy, and the disruption of these functions may result in cell death (Nicotera et al., 1998; Wallace and Starkov, 2000; Szewczyk and Wojtczak, 2002). In in vivo studies performed by Lowenstein et al. (1996) and Hsu et al. (2001), ABA caused an elevation in the concentration of the AST in blood serum. El-Shenawy (2010) performed an in vitro study with isolated rat hepatocytes to compare the toxic action of several insecticides. Among the tested insecticides was ABA, which was used at concentrations of 10 and 100 lM. The results obtained by El-Shenawy (2010) showed a significant increase in ALT and AST leakage when the hepatocytes were incubated with 10 and 100 lM ABA for 30–120 min (final period of sample collection). Necrosis and apoptosis are types of cell death. One evident physiological difference in cells undergoing apoptosis versus necrosis is in the intracellular levels of ATP. Whereas necrotic cell death occurs in the absence of ATP, apoptosis depends on intracel- lular ATP levels (Tsujimoto, 1997). Many key events in apoptosis focus on the mitochondria, including the release of caspase activa- tors (such as cytochrome c), changes in electron transport, loss of mitochondrial transmembrane potential, altered cellular oxida- tion–reduction, and participation of pro- and antiapoptotic Bcl-2 family proteins (Green and Reed, 1998). Thus, in this study, the parameters related to both types of cell death were monitored, allowing the type of cell death triggered by ABA in isolated hepa- tocytes to be distinguished. The release of cytochrome c and caspase 3 activity are steps in determining apoptosis establishment for the intrinsic pathway (Kass et al., 1996; Barros et al., 2003). For both parameters, we have not found significant variation in apoptosis induction in hepato- cytes exposed to ABA. Necrosis is characterized by changes that cause depletion of ATP, disruption of ionic equilibrium, swelling of mitochondria and the cell, and activation of degradative enzymes. These changes result in the disruption of the plasma membrane and loss of proteins, intracellular metabolites and ions (Eguchi et al., 1997; Nicotera et al., 1998; Lemasters et al., 1999). Following microscopic evaluation of Hoechst-propidium-iodide double staining, it was confirmed that ABA induces necrosis, which was initially observed at 60 min in a concentration- and time-dependent manner upon the addition of 75 and 100 lM of ABA and that proadifen stimu- lated this effect. This study indicates that the mechanism of ABA hepatotoxicity involves an effect on mitochondrial bioenergetics and alteration in calcium homeostasis, which leads to a decrease in ATP synthesis with consequent cell death by necrosis (Fig. 8). Furthermore, this study shows that the metabolism of ABA, which is performed by cytochrome P450 in the liver, influences its toxicity. For all vari- ables evaluated, there was an increase in the toxic potential of ABA in the presence of proadifen, indicating that the parent drug has greater potential than the metabolites. Conflict of interest statement The authors declare that there are no conflicts of interest. 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The role of mitochondria and biotransformation in abamectin-induced cytotoxicity in isolated rat hepatocytes 1 Introduction 2 Materials and methods 2.1 Chemicals 2.2 Animals 2.3 Isolation and incubation of hepatocytes 2.4 Oxygen uptake 2.5 Mitochondrial membrane potential 2.6 Cellular ATP content 2.7 Evaluation of cell viability 2.8 Intracellular Ca2+ homeostasis 2.9 Release of cytochrome c 2.10 Caspase 3 activity 2.11 Necrotic cell death 2.12 Statistical analysis 3 Results 3.1 Effects of ABA on the respiration of mitochondria in isolated rat hepatocytes 3.2 Effects of ABA on the mitochondrial membrane potential and ATP levels in isolated rat hepatocytes 3.3 Effects of ABA on cell viability in isolated rat hepatocytes 3.4 Effects of ABA on intracellular Ca2+ homeostasis in isolated rat hepatocytes 3.5 Effects of ABA on cytochrome c release in isolated rat hepatocytes 3.6 Effects of ABA on caspase 3 activity in isolated rat hepatocytes 3.7 Effects of ABA on the induction of necrotic cell death in isolated rat hepatocytes 4 Discussion Conflict of interest statement Acknowledgements References