Abstract
The bioenergetics of cardiac, liver, and kidney mitochondria after 9-day treatment regimen with benzonidazole was studied in rats. The drug was given by oral gavage to adult male Sprague-Dawley rats for 9 consecutive days (100 mg benzonidazole/kg body weight as daily dose). The assayed mitochondrial bioenergetic parameters were the state 4, state 3, respiratory control, efficiency of oxidative phosphorylation, and the activity of the mitochondrial ATP synthase. The results showed that mitochondrial parameters were not altered statistically after in cardiac and kidney mitochondria, but respiratory control in liver mitochondria was statistically increased with benzonidazole treatment. This change was likely due to a slight decrease in state 4 bioenergy metabolism. These results indicate that 9-day benzonidazole treatment regimen had no negative effect on cardiac, liver, and kidney mitochondrial energy metabolism but increased respiratory control in rat liver mitochondria.
Benzonidazole is currently the treatment of choice for Chagas disease (Rodrigues and de Castro 2002), a condition caused by Trypanosoma cruzi infections, affecting several million Latin Americans (Weir 2006), and is one of the leading causes of infectious myocarditis (Feldman and McNamara 2000). Benzonidazole is effective for treating acute stage of Chagas disease, but its effectiveness for treating chronic stage remains uncertain (Viotti et al. 2006). Recently, some investigators (de Souza et al. 2000; Toledo et al. 2004; Garcia et al. 2005; Viotti et al. 2006) have published on the use of benzonidazole treatment in chronic stage of Chagas disease. Given the lack of therapeutic options for Chagas disease, the potential benefits of benzonidazole for chronic treatment of the disease should be carefully studied (Garcia et al. 2005). It is also very important to address whether benzonidazole treatment alone adversely affects the myocardium, because clinical use may result in organ-specific toxicity (Castro and Díaz de Toranzo 1988; Rodrigues and de Castro 2002).
Mitochondria transform and store, at physiological conditions, more than 90% of the cellular energy within the cardiomyocyte (Harris and Das 1991) and represent roughly 25% to 35% of the cell volume within cardiomyocites. For these reasons, cardiac mitochondria take crucial part in molecular/cellular mechanisms of pathophysiological events at heart (Jennings et al. 1978). It is known that cardiac mitochondria are the targets of toxicity for some drugs and chemical agents (Scott et al. 1970; Moreno-Sanchez et al. 1999; Sanchez et al. 2000, 2001a). It was therefore considered important to study whether benzonidazole administration alone could negatively impact cardiac mitochondrial function, compromising normal cellular energy supply for different biological processes at myocardium. Additionally, after benzonidazole administration, mitochondria from various organs may be potential targets of toxicity, as is known for other chemical mitochondrial toxins (Chagas et al. 1992, 1995a; Solem, Henry, and Wallace 1994; Costantini et al. 1995; Da Lozzo, Oliveira, and Carnieri 1998; El-Hassan et al. 2003), therefore the current mitochondrial study was also undertaken on liver and kidney mitochondria. The benzonidazole treatment regimen, used in the present study, was a 9-day treatment regimen (100 mg benzonidazole/kg body weight as daily dose); this treatment regimen was selected because it was tested, with good results, in experimentally infected mice with Trypanosoma cruzi (de Souza et al. 2000), controlling parasite growth, and favoring survival. Mitochondria possess an inner energy-transducing membrane, in which its electron transfer chain catalyzes the downhill transfer of electrons from oxidative substrates to final acceptor O2 and uses this energy to generate a gradient of protons that forces mitochondrial ATP synthase to synthesize ATP from ADP and P i . Mitochondrial bioenergetics is studied by assaying directly the rate of the final transfer of electrons to O2, and information on many other mitochondrial processes can be obtained simply by arranging the mitochondrial incubation conditions (mitochondria at different metabolic states). From this assay, at specific metabolic states (state 4 and state 3; see Materials and Methods for details), it is possible to assess the integrity of a mitochondrial preparation (respiratory control), to calculate the amount of ATP per minute per mg of protein synthesized by mitochondria (the activity of the mitochondrial ATPsynthase), and the efficiency of this process of ATP synthesis (the efficiency of oxidative phosphorylation). Alterations of any of these parameters compromise normal supply of mitochondrial ATP energy for important biological processes, implicating possible cellular, tissue, and organ injuries.
Overall, the current investigation was aimed at studying the mitochondrial bioenergetics of heart, kidney, and liver mitochondria after a 9-day treatment regimen with benzonidazole. The studied mitochondrial bioenergetic parameters were the state 4, state 3, respiratory control, efficiency of oxidative phosphorylation, and the activity of the mitochondrial ATPsynthase.
MATERIALS AND METHODS
Benzonidazole Administration
Male Sprague-Dawley rats, raised in the animal facilities at the Antioquia University, were used in the experiments and were provided during the study at our biophysical laboratory with food and water ad libitum. Benzonidazole (N-benzyl-2-nitroimidazoyl acetamide; Rochagan, Roche) was prepared daily by trituration and suspension of the tablet in distilled water. The therapeutic scheme with benzonidazole was according to de Souza et al. (2000), in which the drug was given by oral gavage to a group of eight rats for 9 consecutive days (100 mg benzonidazole/kg body weight as daily dose). Other group of eight animals (control group) received by oral gavage only distilled water for 9 consecutive days (the same daily volume dose than treated animals). Rats were sacrificed 24 h after the last day of the treatment regimen. All animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals (1996; National Academy Press).
Preparation of Mitochondria and Assay of Mitochondrial Bioenergetic Parameters
All rats were fasted overnight. Heart mitochondria were isolated according to (Mela and Seitz 1979); rat liver mitochondria were isolated according to (Lemeshko 2002); and rat kidney mitochondria were isolated according to (Weinberg, Harding, and Humes 1982). The rates of mitochondrial oxygen consumption were determined by a Clark oxygen electrode in the basal medium 100 mM sucrose, 75 mM KCl, 10 mM HEPES, 0.1 mM EGTA, 10 mM KH2PO4, 1 mM MgCl2, pH 7.4; the oxidative substrate was 4 mM glutamate plus 1 mM malate or 5 mM succinate–2.5 μM rotenone. State 4 was the rate of oxygen consumption when mitochondria were only in the presence of oxidative substrate; state 3 was the rate of oxygen consumption when mitochondria were in the presence of oxidative substrate plus ADP; and the respiratory control (RC) is the ratio state 3/state 4. The efficiency of oxidative phosphorylation (ADP:O) is the ratio ADP/O, where ADP is the amount of added ADP at state 3 (450 nmol ADP), and O (1/2 O2) is the amount of oxygen uptake during state 3; the activity of the mitochondrial ATP-synthase (the ATPsynthase activity) was estimated from state 3 and ADP:O parameters. All experiments were carried out at 30°C using 1 mg of mitochondrial protein/ml. Protein was determined by the biuret method (Gornall, Bardawill, and David 1949). All reagents were of analytical grade and were purchased from Sigma.
Statistical Analysis
All values were means ±SEM of four/eight independent experiments in duplicate, and their statistical significance (p < .05) was evaluated using Student’s t test. Statistical analysis was performed by GraphPad PRISM version 2.0 software (San Diego, CA, USA).
RESULTS
The relative organ weights for heart, kidney, and liver after 9 days of treatment regimen with benzonidazole or water are shown in Figure 1. Relative liver weights were elevated in the drug-treated group compared to controls (Figure 1C ), reflecting possible liver hypertrophy or hepatocytomegaly or both.
In Tables 1 and 2, the mitochondrial bioenergetic parameters of heart and kidney mitochondria, using glutamate/malate or succinate as substrate, after 9 days of treatment regimen with benzonidazole are depicted. There were no statistical differences between drug-treated animals and controls.
In the Figure 2, state 4, state 3, and RC of liver mitochondria, using succinate as substrate, after 9 days of treatment regimen with benzonidazole are presented. After this drug treatment regimen, state 4 (Figure 2A ) showed non statistical decrease (9.2%), state 3 (Figure 2B ) was practical equal between groups (1.9%), and the treated RC (Figure 2C ) was statistical different (11.8%) with respect to its control.
In the Figure 3, state 4, state 3, and RC of liver mitochondria, using glutamate/malate as substrate, after 9 days of treatment regimen with benzonidazole are shown. After this treatment regimen, state 4 (Figure 3A ) showed nonstatistical decrease (9.5%), state 3 (Figure 3B ) was practical equal between groups (3.8%) and the treated RC (Figure 3C ) was not statistical different (11.5%) with respect to its control.
DISCUSSION
In Figure 1, it was shown that only relative liver weights were altered after the 9-day benzonidazole treatment regimen; this ratio was 28% greater in treated rats than in control ones, indicating that the liver is the site of potential toxicity for this drug given repeatedly.
Tables 1 and 2 showed that the 9-day benzonidazole treatment regimen did not decrease ATPsynthase activity and the ADP/O ratio, using glutamate/malate or succinate as substrate, of heart and kidney mitochondria; i.e., heart and kidney mitochondria after this benzonidazole treatment regimen were able to synthesize ATP with a rate and an efficiency sufficient to normally preserve the mitochondrial energy supply for heart and kidney cells. These tables also showed that state 4, state 3, and RC from control and treated groups, for heart and kidney mitochondria, are practically equal. Taken together, all these data indicated that mitochondrial bioenergetics of heart and kidney mitochondria was not altered after the current benzonidazole administration, warranting normal supply of mitochondrial cellular energy for the biological processes, taking place at heart and kidney cells after this benzonidazole treatment regimen. The above-mentioned results for heart mitochondria correlated with the results obtained by de Souza et al. (2000), using the same 9-day benzonidazole treatment regimen. Their results showed that the 9-day benzonidazole treatment regimen alone did not cause myocardium cellular lesions tested by both creatine kinase and its cardiac creatine kinase isoenzyme plasma levels (enzymes that are useful indicators of myocardium cellular lesions).
When the liver mitochondria were studied, ATPsynthase activity and the ADP/O ratio, using glutamate/malate or succinate as substrate, were practical equal upon 9-day benzonidazole treatment (data not show); i.e., liver mitochondria after this benzonidazole administration were able to synthesize ATP with a rate and an efficiency sufficient to preserve normal mitochondrial energy supply for liver biological processes. The data, shown in Figure 2C , display a statistical slight increase in liver RC, using succinate as substrate, after the 9-day benzonidazole treatment regimen (11.8% of statistical difference). Figure 2B shows that the means of liver state 3, using succinate as substrate, from control and treated groups are practically equal (1.9% of not statistical difference). Figure 2A shows that the mean of liver state 4, using succinate as substrate, has small tendency to decrease upon 9-day benzonidazole treatment (9.2% of non statistical difference). Thus, liver state 4 is the main cause in the statistically slight increase of liver RC (RC = state 3/state 4), using succinate as substrate, upon this benzonidazole treatment. It is important to point out that all mean tendencies of liver state 4, liver state 3, and liver RC were similar when it was used succinate or glutamate/malate as substrate (Figures 2 and 3), upon 9-day benzonidazole treatment; but only the mean tendency for liver RC, using succinate as substrate, was statistical different. When mitochondria are respiring using glutamate/malate or succinate, their respiratory chains are feeding with electrons from Krebs cycle to complex I or from succinate to complex II, respectively. Glutamate/malate respiring mitochondria are more complex system than succinate respiring mitochondria, because in the first case the Krebs cycle works too. It was possible that the statistical slight difference for liver succinate RC (11.8%; p < .03), after this benzonidazole treatment regimen, became not statistical using glutamate/malate as substrate (11.5%; p < .06), due to the above mentioned additional complexness. Taken together, all these data indicated that mitochondrial bioenergetics of liver mitochondria was not negatively altered after the 9-day benzonidazole treatment regimen, warranting normal supply of mitochondrial cellular energy for the biological processes taking place at liver cells after this benzonidazole treatment regimen.
Overall, this work shows a lack of toxicity of the 9-day benzonidazole treatment regimen upon the bioenergetics of heart, kidney, and liver mitochondria; however, clinical use of benzonidazole may result in organ-specific toxicity (Castro and Díaz de Toranzo 1988, Rodrigues and de Castro 2002), pointing out the necessity for carrying out further studies to find cellular targets of benzonidazole toxicity.
Footnotes
Figures and Tables
This work was supported by the financial contribution of COL-CIENCIAS, grant no. 1118-04-16552, and the National University of Colombia. The author wish to thank Dr. Armando Galeano, Coordinador de Vectores-Dirección Nacional de Salud de Antioquia, for his generous gift of the benzonidazole Rochagan® tables.