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.