The effect of antibiotics on the mitochondrial function in rat skeletal muscle

Abstract

BackgroundMitochondria are the main energy producing organelle of the cell through oxidative phosphorylation (OXPHOS) and the electron transport system, and major sources of reactive oxygen species (ROS) result from superoxide (O2−) and hydrogen peroxide (H2O2). Accumulation of ROS has the potential to harm macromolecules such as DNA and RNA. How antibiotics contribute to oxidative damage in cells have not been sufficiently investigated, although Kohanski et al. (1) have proposed a mechanism of how bactericidal antibiotics increases O2− production causing an excessive production of hydroxyl radical (OH−) via the Fenton reaction. This study has therefore investigated the effect of clarithromycin, trimethoprim and penicillin on the mitochondrial respiration and H202 production in rat skeletal muscle to see if our findings correlate with Kohanski's proposal.MethodAn approach combining high‐resolution respirometry and fluorometric measurement of H2O2 production by the Oroboros Oxygraph 2K instrument was used. Skeletal muscle tissue from mice (C57BL/6JRJ) was prepared and permeabilized using saponin stock solution. A stepwise multiple substrate‐uncoupler‐inhibitor titration (SUIT) protocol was used to analyze the oxidative phosphorylation, the mitochondrial respiratory control, and H2O2 production. The drug concentrations of antibiotics were equivalent to those from clinical use. 6 muscle samples were exposed to each antibiotic and 6 were used as control. The software Datlab was used to calculate mitochondrial oxygen flux per mass [pmol/(s*mg)] and the H2O2 production using a fluorescent probe.ResultsOur findings showed that clinical doses of clarithromycin increased the mitochondrial respiration by 3.62 pmol, SD= ±4.71 compared to control which showed a decrease in respiration by −0.84 pmol, SD= ±1.16 (p‐value = 0.063). There were not observed any significant alterations in H202 production with the addition of clarithromycin as the H2O2 production increased by 0.52 pmol, SD= ±0.75 compared to control 0.011 pmol, SD= ±0.037 (p‐value = 0.164). Trimethoprim and penicillin on the other hand caused a decrease in mitochondrial respiration by −1.11 pmol SD= ±3.01 and −4.65 pmol SD= ±25.09 compared to their respective control −0.46 pmol SD= ±1.27 and 3.46 pmol SD= ±5.38, (p‐value = 0.706). No significant findings were observed with the H2O2 production.ConclusionOur findings suggest that clarithromycin possibly increases the mitochondrial respiration and leads to an increased production of O2−. Clarithromycin did increase the H202 production slightly. This study does not finally elucidate that antibiotics contribute to the formation of ROS, but can on the other hand assist the further understanding of drugs and mitochondrial dysfunction.Support or Funding InformationEdon Morina1, Vanja Cejvanovic1, Laura Kofoed Kjær1, Henrik Enghusen Poulsen1, Søren Nielsen21Q7642, Department of Clinical Pharmacology, Bispebjerg Hospital; 2Department of Health Science and Technology Aalborg University