In vitro cytotoxic and ultrastructural effects of selected carboxylic ionophores on cardiac and skeletal myoblasts

Abstract

Carboxylic ionophores are polyether antibiotics that are extensively used in production animals for the control of coccidiosis and to promote growth and feed efficiency. Unfortunately, due to feed mixing errors and extra-label use, cases of ionophore toxicosis occur and primarily affect cardiac and skeletal muscles. Ionophores form dynamically reversible complexes with cations and facilitate their movement across biological membranes, affecting the ion homeostasis of cells and disrupting their normal physiological functions. The aim of the study was to determine the cytotoxicity, subcellular changes and ultrastructural effects caused by the three different antibiotics, monensin, salinomycin and lasalocid on cardiac (H9c2) and skeletal (L6 and C2C12) myoblasts in vitro. The cytotoxicity of each ionophore over a 72-h period was determined using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) viability assay. Immunofluorescent and immunocytochemistry techniques were used to investigate the effect of the ionophores on the microfilament and microtubule networks, as well as on the intermediate filaments desmin and synemin. Finally, electron microscopy was used to determine the ultrastructural effects of ionophore exposure. Monensin induced the highest cytotoxicity of the three ionophores, with EC50s in the low nanomolar range after 48 and 72 h exposure, followed by salinomycin and lasalocid. Myoblasts exposed to the different ionophores had similar morphological changes, with cytoplasmic vesicles filling the entire myoblast as well as myoblasts ‘rounding off’ and detaching from their surroundings. The microfilament and microtubule networks showed minimal to moderate disruption depending on the cell line, ionophore, ionophore concentration and exposure time. Disruption to the intermediate filament network was observed after monensin and salinomycin exposure, resulting in the aggregation of desmin around the nuclei of affected myoblasts. However, the synemin network was the least affected. Ultrastructural changes included the accumulation of electron-lucent vesicles within the cytoplasm, mitochondrial and chromatin condensation as well as myoblasts with apoptotic and necrotic features. In conclusion, of the carboxylic ionophores tested monensin was the most cytotoxic in vitro. Furthermore, ionophore exposure resulted in the disruption of the cytoskeletal networks, especially desmin filaments, which could contribute to the myofibrillar degeneration and necrosis seen in the skeletal muscles of animals suffering from ionophore toxicosis. Finally, cytoplasmic vesiculation and mitochondrial condensation was the most prominent ultrastructural changes that occurred as a result of ionophore exposure in vitro, with both apoptotic and necrotic myoblasts present.