Maduramicin is a polyether ionophore

Maduramicin is a polyether ionophore antibiotic that has the smallest margin of safety between the maximum authorized dosage and the minimum toxic concentration. Clinical signs of maduramicin intoxication are similar to that of other ionophores, often causing feed refusal, anorexia, respiratory distress, lethargy, ataxia, recumbency, and sudden death (Mcnaughton, 1991; Shlosberg et al., 1992, Shlosberg et al., 1997). Heart and skeletal muscles have been identified as the main target organs of maduramicin toxicity (Todd et al., 1984; Novilla and Folkerts, 1986; Anadon and Martinezlarranaga, 1991; Novilla and Todd, 1991; Van Vleet et al., 2002; Matsuoka et al., 1996). Toxic levels of maduramicin have been implicated in the death of various animal species, including chickens, cattle, pigs, and sheep, mainly due to cardiac and/or respiratory failure (Fourie et al., 1991; Shlosberg et al., 1997). Another ionophore antibiotic, monensin, affects cation transport across the cell membrane, which in turn increases the influx of Ca2+ and thereby causes cell membrane disruption and cell death (Sandercock and Mitchell, 2003). In a previous study, we showed that maduramicin is toxic to mouse C2C12 myoblasts and human rhabdomyosarcoma RD and Rh30 cells by inhibiting cell proliferation and inducing apoptosis (Chen et al., 2014). Nevertheless, to date, the toxicity and the underlying mechanism of action of maduramicin in chicken myocardial cells remain unclear. In the present study, we investigated the toxicity and mechanism of action of maduramicin in primary chicken myocardial cells.
Materials and methods
Results
Discussion Maduramicin is approved for use in the USA, European Union, China, and many other countries as a coccidiostat for broiler chickens and turkeys, with a recommended level of 5 mg/kg in feed. In the USA, >90% of broiler chickens and about 75% of cattle marketed per year have consumed ionophore dihydrofolate reductase inhibitor antibacterial during their lifetime (Novilla, 2012). Compared to other ionophore compounds, maduramicin has a small safety margin and frequently causes intoxication of target and nontarget animals via overdosage, misuse, or interactions with other medications (Novilla, 1992; Oehme and Pickrell, 1999; Sharma et al., 2005; Shimshoni et al., 2014). A previous report has shown that intoxication is mainly characterized by the degeneration and necrosis of cardiac and skeletal muscles (Novilla, 1992). However, the exact mechanism by which maduramicin induces toxicity remains unclear. Hence, the aim of the present study was to investigate the cytotoxic effect of maduramicin and to elucidate its underlying cellular and molecular mechanisms using primary cultures of chicken myocardial cells as an in vitro model. To characterize the in vitro cytotoxic effect of maduramicin, we first studied the cell morphology and viability of chicken myocardial cells exposed to maduramicin. The results showed that cellular atrophy and vacuolization occurred in all treatment groups and all time intervals. Cell detachment from the substrate was also seen, probably as a consequence of the maduramicin-induced apoptosis or other cellular damage. Exposure to maduramicin resulted in a concentration- and time-dependent decrease in cell viability, i.e., prolonged exposure to low concentration or acute exposure to relatively high concentrations caused cell death. These results agree with previous studies (Scherzed et al., 2013; Antoszczak et al., 2014; Cybulski et al., 2015). Our previous study has shown that maduramicin imparts its cytotoxic effects on mouse myoblasts C2C12 cells and human rhabdomyosarcoma cells (RD and Rh30) by inducing apoptosis (Chen et al., 2014). Other ionophore antibiotics such as salinomycin and monensin also trigger apoptosis (Park et al., 2003; Zhou et al., 2013). We therefore hypothesized that the cytotoxicity of maduramicin in chicken myocardial cells is also associated with the induction of apoptosis. The externalization of phosphatidylserine (PS) from the inner leaflet of the plasma membrane is characteristic of early apoptosis in cells (Shiratsuchi et al., 1998). Annexin V-positive/PI staining and DAPI staining showed that apoptosis was induced in a concentration-dependent manner when cells were treated with maduramicin. These results are in agreement with our obtained findings of transcriptome analysis (Gao et al., 2017).