Belosludtsev 2018 MiP2018
Bedaquiline (BDQ) and delamanid have recently been approved for the treatment of multidrug- and extensively drug-resistant tuberculosis. The antibacterial effect of these compounds has been well studied. The mechanism of BDQ antibacterial effect is based on the inhibition of bacterial F1/FO ATP-synthase, which leads to suppression of the process of oxidative phosphorylation, poor ATP production and, eventually, cell death. Delamanid inhibits the biosynthesis of mycolic acids of cell wall of Mycobacterium tuberculosis .
An important step in the introduction of a new antibacterial preparation into medical practice is the study of its influence on the eukaryotic cell, in order to reveal its possible side effects in the human and animal organisms. At the same time, data on the interaction of BDQ and delamanid with cells of eukaryotic organisms are scarce: these drugs were introduced into practice not a long time ago and in quite a hasty manner . The available literature data indicate that the influence of BDQ on eukaryotic cells is based on, at least, two mechanisms: (1) its effect on cell energetics and (2) its effect on the operation of membrane enzymes and ion channels. In this connection, of special interest is the question of how BDQ and delamanid interact with mitochondria.
Mitochondria were isolated from the liver of Wistar rats by differential centrifugation. The rate of oxygen consumption of rat liver mitochondria was measured polarographically with a Clark-type gold electrode (Oroboros Instruments, Oxygraph-2k, Austria) at 25 °C under continuous stirring. The swelling of mitochondria (0.4 mg/mL) was measured as a decrease in A540 in a stirred cuvette at room temperature (~22 °C) using a USB-2000 spectroscopy fiber-optic system (Ocean Optics, USA). The release of cytochrome c from mitochondria was determined using western-blot analysis.
The effect of BDQ on the functional state of rat liver mitochondria was evaluated by the rate of mitochondrial respiration with glutamate/malate (substrates of Complex I of the respiratory chain) or succinate (a substrate of Complex II) in the presence of rotenone . 10-20 µM BDQ had little effect on the succinate-fueled respiration (less than a 10% decrease) in all the functional states (JROX, VP, JL and JE). However, raising BDQ concentration to 50 µM resulted in a marked inhibition of mitochondrial respiration: the rates of respiration in OXPHOS- and ET(DNP)-states were lowered by 40 and 44% of control, respectively. The parameter of respiratory control decreased by 20%. At the same time, the efficiency of ATP synthesis estimated as ADP/O ratio remained practically the same (1.90 ± 0.09 in control measurements and 1.82 ± 0.11 in the presence of BDQ). The time of phosphorylation, however, increased almost 2-fold (from 109 ± 5 s to 208 ± 15 s) when BDQ was added. With NAD-dependent substrates (2.5 mM glutamate and 2.5 mM malate), the effect of BDQ on mitochondrial respiration was different from the effect observed with succinate. 50 µM BDQ caused a slight inhibition of JP and JE respiration (approximately, by 10-15% of control). This was accompanied by a decrease of the respiratory control level by less than 1.
The inhibitory effect of BDQ on ATP synthesis seems to be associated with the suppression of the functioning of the mitochondrial respiratory chain BDQ practically did not affect the activity of Complex III and Complex IV. At the same time, it suppressed the activity of Complex II, which was especially evident when the combined activity of Complex II + III was assessed (a 60% inhibition by 50 µM BDQ). These results indicate that BDQ hinders the access of coenzyme Q to the respiratory complexes.
In contrast to BDQ, delamanid caused a marked inhibition of mitochondrial respiration using substrates of Complex I and II of the respiratory system. The rates of respiration in OXPHOS- and ET-states in the presence of 20-100 µM delamanid were lowered by almost 2-fold compared with the control ones.
BDQ and delamanid can result in the inhibition of H2O2 production by rat liver mitochondria. The effect of different concentrations of these compounds on ROS production fits well in the scheme of action of antioxidants, which exhibit antioxidant properties at low concentrations but exert a prooxidant effect at higher concentrations.
The development of tuberculosis is known to be accompanied by the opening of MPT pore in mitochondria of infected cells. BDQ inhibited swelling of rat liver mitochondria induced by 50 µM Ca2+ in the presence of 1 mM Pi. The inhibition was dose-dependent, with 50 µM BDQ almost completely suppressing the swelling. When mitochondria are preincubated with BDQ, a significant part of the organelles does not undergo structural changes upon the Ca2+/Pi-induced opening of the pore. In addition, As it is known, the opening of MPT pore is one of the major pathways for the induction of cell death, with the mechanism being related to the release of proapoptotic proteins from the organelles (cytochrome c, AIF etc.). BDQ prevents the release of cytochrome c from mitochondria.
At the same time, delamanid stimulated swelling of rat liver mitochondria induced by 50 µM Ca2+ in the presence of 1 mM Pi. Thus, it can be concluded that delamanid in the used concentrations is able to exert a toxic effect on mitochondria of rat liver.
The results obtained in the present work indicate a complex effect of BDQ on mitochondria. BDQ causes inhibition of both physiologically relevant and pathology-related mitochondrial processes. It is important to note that the data of most of the BDQ tests conducted by now indicate that BDQ has no toxic effects on normal eukaryotic cells, only affecting bacterial and abnormal (both tuberculosis-infected and cancer) eukaryotic cells [4,5]. In this respect, the results of our work obtained on the suspensions of mitochondria of healthy animals confirm those data and allow us to conclude that BDQ can protect normal cells from the development of oxidative stress and cell death.
Labels: MiParea: Respiration, Pharmacology;toxicology
Organism: Rat Tissue;cell: Liver Preparation: Isolated mitochondria
Pathway: N, S HRR: Oxygraph-2k
Affiliations and support
Belosludtsev KN(1,2), Belosludtseva NV(2), Talanov EYu(2), Starinets VS(1), Dubinin MV(1)
- Mari State Univ, Yoshkar-Ola
- Inst Theoretical Experimental Biophysics RAS, Pushchino; Russia – firstname.lastname@example.org
- The work was supported by grants from the Russian Foundation for Basic Research and Government of Moscow region (17-44-500584 to BKN, 18-34-00297 to BNV) and the Ministry for Education and Science of Russian Federation (6.5170.2017/8.9 to BKN).
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