Ultrastructural changes in cardiac and skeletal myoblasts following in vitro exposure to monensin, salinomycin, and lasalocid.

Carboxylic ionophores are polyether antibiotics used in production animals as feed additives, with a wide range of benefits. However, ionophore toxicosis often occurs as a result of food mixing errors or extra-label use and primarily targets the cardiac and skeletal muscles of livestock. The ultrastructural changes induced by 48 hours of exposure to 0.1 µM monensin, salinomycin, and lasalocid in cardiac (H9c2) and skeletal (L6) myoblasts in vitro were investigated using transmission electron microscopy and scanning electron microscopy. Ionophore exposure resulted in condensed mitochondria, dilated Golgi apparatus, and cytoplasmic vacuolization which appeared as indentations on the myoblast surface. Ultrastructurally, it appears that both apoptotic and necrotic myoblasts were present after exposure to the ionophores. Apoptotic myoblasts contained condensed chromatin and apoptotic bodies budding from their surface. Necrotic myoblasts had disrupted plasma membranes and damaged cytoplasmic organelles. Of the three ionophores, monensin induced the most alterations in myoblasts of both cell lines.

Cytoprotective effect of silybin against lasalocid-induced toxicity in HepG2 cells.

Lasalocid is an ionophore coccidiostatic agent frequently used in poultry. Its extensive use causes the formation of residues in edible tissues and eggs which may pose a risk to consumers. Silybin is the main compound extracted from the herb milk thistle Silybum marianum and its hepatoprotective effect has been reported in literature. The aim of the study was to compare lasalocid and silybin cytotoxic effects followed by their combined use in HepG2 cell line. A cytoprotective effect resulting from the interaction of both pharmacologically active substances was measured. In this study, an MTT test, coomassie brillant blue binding test, and LDH release test determined the effective concentration (EC50) of the compounds. The isobolograms and combination index were used to assess the nature of interaction. The lowest EC50-value for lasalocid was established via the MTT test. This study revealed a lack of silybin cytotoxic effect on the cells. Co-actions of the two drugs led to a significant decrease of lasalocid cytotoxicity. The isobolograms and combination index showed a remarkable antagonistic effect in the course of silybin and lasalocid interaction. The results indicate that silybin revealed a cytoprotective effect when incubated with lasalocid since its cytotoxic impact on HepG2 cells has been significantly diminished.

Toxicity of the ionophore antibiotic lasalocid to soil-dwelling invertebrates: avoidance tests in comparison to classic sublethal tests.

Lasalocid is a veterinary ionophore antibiotic used for prevention and treatment of coccidiosis in poultry. It enters the environment with the use of contaminated manure on agricultural land. Despite its extensive use, the effects of lasalocid on non-target soil organisms are poorly explored. We used classical subleathal ecotoxicity tests to assess the effects of lasalocid on earthworms (Eisenia andrei) and isopods (Porcellio scaber) and compared the results with tests using avoidance behaviour as the endpoint. The results showed that avoidance is a much more sensitive endpoint. For earthworms, EC50 for avoidance (12.3 mg kg(-1) dry soil) was more than five times lower than EC50 for reproduction (69.6 mg kg(-1) dry soil). In isopods the sensitivity of the behavioural response test was even higher. While the highest lasalocid concentration 202 mg kg(-1) had no significant effects on isopod growth or survival, already the lowest used concentration in the behavioural assay (4.51 mg kg(-1)) caused significant impact on isopod behaviour. Using the avoidance test results for calculating the predicted no-effect concentration (PNEC) of lasalocid to soil invertebrates, the value is close to the predicted environmental concentration (PEC). This indicates that the use of lasalocid-contaminated manure could potentially impair the habitat function of agricultural soils.

The protective effect of silybin against lasalocid cytotoxic exposure on chicken and rat cell lines.

Lasalocid, an ionophore coccidiostat, extensive use implies a risk of toxicological impacts. Protective effects of silybin, a herbal compound of Silybum marianum, are reported elsewhere. The aim of this study was to compare effects of the combined use of lasalocid and silybin in chicken hepatoma cells (LMH) and rat myoblasts (L6) cell lines cultures. The cytoprotective effect resulting from an interaction of both pharmaceuticals was measured with the help of MTT reduction and, coomassie brilliant blue binding (CBB) and LDH release assays. Isobolography and the combination index (CI) estimated the nature and scale of interaction. In all performed tests, the lowest lasalocid EC50-values were obtained for chicken hepatocytes. In the rat myoblasts cultures, the lowest lasalocid EC50-values were found with LDH test. Simultaneously, a lack of silybin cytotoxic effect was proven for the studied cell lines. An interaction between both substances led to a considerable decrease of lasalocid cytotoxicity. The isobolograms and combination index showed a significant antagonistic nature of silybin effect in the course of lasalocid cytotoxicity. It is concluded that the mechanism of cytoprotection results from complex reaction at biochemical and biophysical endpoints during chicken hepatocytes and rat myoblasts cell lines exposure to silybin and lasalocid co-action.

Tissue Doppler imaging and 2-dimensional speckle tracking of left ventricular function in horses exposed to lasalocid.

BACKGROUND: Tissue Doppler imaging (TDI) and 2-dimensional speckle tracking (2DST) can quantify left ventricular (LV) function in horses. OBJECTIVES: To evaluate LV function by TDI and 2DST in horses with myocardial dysfunction after accidental ionophore intoxication. ANIMALS: Sixty-seven horses exposed to lasalocid in feed. METHODS: Prospective study. Horses were included in the study if a full cardiac examination was performed, consisting of determination of cardiac troponin I (cTnI), electrocardiography, and echocardiography. By TDI, radial systolic velocity and strain were measured. By 2DST, circumferential (SC) and radial (SR) strain at papillary muscle and chordal level and longitudinal (SL) strain were measured. RESULTS: Twenty horses showed signs of myocardial injury. Forty-nine examinations were performed on these horses between day 30 and 490 after suspected onset of exposure. Five horses had increased cTnI and ventricular tachycardia and 15 had increased cTnI without ventricular tachycardia. Horses with mild myocardial damage showed few significant differences compared with a control group. Horses with severe myocardial damage showed severely decreased TDI, 2DST and fractional shortening measurements (P < .05), indicating impaired LV function. Long-term follow-up of 2 surviving horses demonstrated full recovery in 1 horse and permanent myocardial fibrosis in the other. The lowest measurements per horse (n = 20) for all TDI measurements, SL, SR at chordal level, and FS correlated significantly with maximal cTnI (P < .05). Over all examinations (n = 49), TDI and 2DST measurements correlated well with FS (P < .05). CONCLUSIONS AND CLINICAL IMPORTANCE: The TDI and 2DST measurements allowed accurate detection and quantification of LV dysfunction in horses exposed to lasalocid.

Lasalocid awareness and sampling in Scotland.

Lasalocid is an ionophore antibiotic extensively used as a coccidiostat in poultry production. Lasalocid should not be fed to egg-laying hens as it accumulates in the eggs, and residues have often been found in eggs. Other ionophores are toxic to humans, but the exact level of lasalocid toxicity to humans has not been established. Approximately 250 egg samples were analysed for lasalocid each year from the 10 billion eggs consumed annually in the UK. A census of the 32 Scottish Local Authority Environmental Health Departments assessed awareness of lasalocid residues in eggs, and the results indicated that awareness of lasalocid was very low and no local authorities tested for lasalocid. The example of lasalocid revealed weaknesses in the current sampling regime surrounding foods of animal origin. Conclusions are drawn that central government should raise awareness within local authorities and provide financial support on local authority sampling to achieve proper representation.

Sorption and degradation in soils of veterinary ionophore antibiotics: monensin and lasalocid.

Monensin and lasalocid are polyether ionophores commonly used in the beef and poultry industries for the prevention of coccidial infections and promotion of growth. These ionophores can exhibit higher toxicity than many other antibiotics; thus, evaluating their fate in the environments associated with concentrated feed operations is important. Sorption of monensin and lasalocid was measured in eight soils of varying physiochemical composition. Organic carbon-normalized sorption coefficients (log Koc) ranged from 2.1 to 3.8 for monensin and from 2.9 to 4.2 for lasalocid and were inversely correlated to equilibrium soil-solution pH. Degradation of lasalocid and monensin in two contrasting soils with and without manure amendment was measured in moist soils at 23 degrees C and 0.03 MPa moisture potential. The half-life of both compounds in the fresh nonsterile soils was less than 4 d, for which monensin degraded slightly faster than lasalocid. Fresh liquid manure amendments did not significantly alter degradation of either compound. Based on parallel 60Co-sterilized soil experiments, some abiotic degradation of monensin was apparent, whereas lasalocid only degraded in the presence of microbes. Analysis of beef-derived lagoon effluent used for irrigation confirmed that monensin can be present at low-ppb to low-ppm concentrations in the aqueous and suspended solids fractions, respectively; however, subsequent analysis of drainage water in a nearby ditch suggested that attenuation by soil after land application will greatly reduce the amount entering surface waters.

Lasalocid toxicosis is inadequately quantified for horses.

The current estimate of LD50, 21.5 mg/kg BW, for lasalocid in horses is based on an analysis of 8 data from 4 horses that died at dose levels of 15, 21, 22 and 26 mg/kg. This analysis neglected 14 data from another 6 horses that survived at dose levels of 5, 10,14, 18, 19, 25, 29 and 50 mg/kg, and so was biased by selection of data. An examination of all the data indicates they are insufficient to determine the LD50. In contrast, the whole data set suggests a lowest toxic dose of 15 mg/kg, although this estimate was based on only 1 affected animal in 8 tests from 5 to 15 mg/kg in an unbalanced experiment and thus needs confirmation.

In-vitro activities of paromomycin and lasalocid evaluated in combination against Cryptosporidium parvum.

Using a chemiluminescence immunoassay, paromomycin and lasalocid were shown to inhibit Cryptosporidium parvum growth in Madin-Darby canine kidney cells in a concentration-dependent manner. The median effective concentrations (EC50s) for paromomycin and lasalocid were 1184 mg/L and 0.4 mg/L, respectively. Neither drug was cytotoxic to host cells at concentrations up to five times their EC50s. Drug combination studies were conducted and the resulting data were analysed by the median-effect principle and combination index method. Statistically significant synergy was observed when combinations of paromomycin and lasalocid were used at ratios of 5000:1 and 2500:1. A possible mechanism for synergy is discussed.

Selective neurotoxicity induced by the ionophore lasalocid in rat dissociated cerebral cultures, involvement of the NMDA receptor/channel.

An in vitro model of dissociated cerebral cultures, prepared from prenatal 15-16-days rat fetuses, was used to further characterize the neurotoxic effects caused by the antibiotic ionophore lasalocid-X-537A. The damage caused by lasalocid (1-2 microM, 2-4 hr) included swelling of perikarya, followed by cytolysis of most neurons present in the cultures. The neuronal damage was dose-dependent, noticeable at concentrations above 0.5 microM, and was more pronounced in established cultures (14 days in vitro-DIV) than in younger ones (7 DIV). Unlike neurons, no damage was observed in glia and other non-neuronal cells present in the cultures by exposure to 2 microM lasalocid. Moreover, the drug was not toxic for cultures of rat astrocytes and C6 glioma cells. Another calcium ionophore A-23187 (calcimycin, 1 microM), destroyed both neuronal and non-neuronal cells within 1 hr. Ca2+ influx was increased by 140% in cultures exposed to lasalocid (1.5 microM). The lasalocid neurotoxic effects were neither inhibited by 10 microM nimodipine (a calcium channel antagonist) nor by 10 microM 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX)(a non-N-methyl-D-aspartic acid (NMDA) receptor antagonist), but were exclusively blocked by 10 microM MK-801 (a non-competitive NMDA receptor/channel antagonist). The neurotoxicity induced by lasalocid was further confirmed by measurements of lactate dehydrogenase (LDH) released into the media. Lasalocid (1.5 microM) induced the release of both LDH and arachidonic acid (AA) (by 8 and 4 fold of control values, respectively), and this was blocked by MK-801 but not by CNQX. These results are in according with the observations that activation of calcium influx through the NMDA receptor leads to activation of phospholipase A2 (PLA2) and release of AA. In contrast, MK-801 did not block the release of either LDH or AA mediated by the calcium ionophore A-23187 (1 microM) in these cultures. [3H]-MK-801 binding to washed rat cortical membranes, a measure of direct interaction with the NMDA receptor/channel complex, was not affected by lasalocid either alone or in the presence of glutamate and glycine. [3H]-D-aspartate release, a measure of excitatory amino acid (EAA) secretion mediated by NMDA receptor activation, was increased by lasalocid and could be blocked by MK-801. These observations suggest that lasalocid induces selective neurotoxicity, which involves the NMDA receptor/channel complex, possibly indirectly, resulted in elevated intracellular Ca2+ levels and the subsequent glutamate or aspartate release.

The influence of ionophores on the duration of xylazine-ketamine anestheisa and the hepatic cytochrome P-450 content of broiler chickens.

This study examined the effects of feeding monensin, lasalocid or salinomycin (at 1x and 2x therapeutic levels for 20 d) on the duration of xylazine+ketamine sleep time and the hepatic cytochrome P-450 content in broiler chickens of different age groups (3- and 6-w-old). The 3-w-old birds fed 120 ppm salinomycin had a reduced xylazine+ketamine sleep time as compared to control (no drug). The 6-w birds had a sleep time similar to the controls. Associated with ionophore feeding was a trend toward P-450 induction when compared to controls. Increased P-450 content was statistically significant in the 3-w birds fed 90 ppm lasalocid and in the 6-w birds fed 200 ppm monensin. Ionophore administration above recommended levels gave conflicting results for duration of xylazine+ketamine sleep time and hepatic cytochrome P-450 levels. The isozymes of cytochrome P-450 responsible for xylazine and ketamine metabolism may be different from those induced by the ionophores. There was no significant age-related difference in ionophore effects on cytochrome P-450 content.

Pathologic changes associated with experimental lasalocid and monensin toxicosis in cattle.

The acute toxicity of lasalocid and monensin was studied in 36 Holstein steers. The cattle were given (orally) a single dosage of lasalocid (1, 10, 50, or 100 mg/kg of body weight) or monensin (25 mg/kg of body weight) or rice hulls. Animals were observed once a day until they died or were euthanatized at 32 days after the dose was given. All cattle were necropsied. Heart, kidney, adrenal gland, liver, spleen, pancreas, lungs, brain, sciatic nerve, skeletal muscle, small intestine, large intestine, and rumen tissue sections, stained with hematoxylin and eosin, were studied microscopically. Lasalocid was lethal at dosages of 50 and 100 mg/kg, and monensin was lethal at the dosage given (25 mg/kg). Cattle dying of lasalocid and monensin toxicoses had gross and microscopic lesions consistent with cardiomyopathy. Dilated heart or petechial and ecchymotic hemorrhages were observed with both drugs. Microscopically, multifocal areas of myocyte necrosis were observed. Those cattle that died within 3 days of dosing with either drug had a marked degranulation of pancreatic acinar cells. Changes were not observed in any other tissues.

Lasalocid toxicity in cattle: acute clinicopathological changes.

Thirty-six steers (148 to 500 kg) divided into six equal groups were used in a toxic syndrome study of lasalocid and monensin given as a single oral dose. One group was given a placebo, a second group received monensin (25 mg/kg body weight) and the other four groups received lasalocid at 1, 10, 50 or 100 mg/kg body weight (bw). No toxic signs developed in cattle given placebo or lasalocid at 1 or 10 mg/kg bw dose. The earliest toxic signs were muscle tremors, tachycardia and rumen atony. After 24 h, the cattle were dehydrated, anorectic and had diarrhea. Deaths occurred between d 1 and 22.5 in the groups receiving lasalocid at 50 and 100 mg/kg bw and monensin. Altered values in blood leucocytes, erythrocytes, hemoglobin, hematocrit, total protein, albumin, creatinine, urea nitrogen, total bilirubin, creatine kinase, lactic dehydrogenase, calcium, chloride and inorganic phosphate occurred 1 d after dosing: urine pH and specific gravity also changed 1 d after dosing. Maximum changes occurred at d 3. Most of the changes were indicative of dehydration rather than specific organ damage.

The action of salinomycin-Na and lasalocid-Na on chloroquine- and mepacrine-resistant line of Plasmodium berghei K 173-strain in Wistar rats.

Salinomycin-Na and lasalocid-Na, two ionophorous antibiotics known for their anticoccidial activity, exhibit in vivo blood schizontocidal action on the Plasmodium berghei Keyberg 173 RC/M line that has a high level of resistance to chloroquine and mepacrine. Salinomycin was found to have a greater effect than lasalocid on asexual stages of this line. Trophozoites and schizonts were no longer found after a single dose of 20 mg/kg or five doses of 1.25 mg/kg of salinomycin whereas a single dose of 40 mg/kg or five doses of 20 mg/kg of lasalocid showed no marked effect on parasitaemia within 96 h of starting treatment in rats. Some toxicological data show that lasalocid, however, is better tolerated in domestic animals than salinomycin. Early morphological changes in asexual blood stages were membrane-coiling in the cytoplasm followed by vacuolization and disruption of the cell membrane or pellicle after treatment with both compounds. In particular mature schizonts were totally destroyed showing enormously large vacuoles. Toxicological data and blood schizontocidal activity indicate the narrow safety margin in P. berghei infected rats, and place salinomycin in the 'markedly toxic' group of antimalarial compounds.

Preliminary studies on lasalocid toxicosis in cattle.

Polyether antibiotics are gaining widespread use in the feedlot cattle industry. One such investigational antibiotic, Lasalocid, was studied at oral doses of 25, 50, 75, 100 and 125 mg/kg to determine effects of toxicosis in six cattle. Deaths occurred at 100 and 125 mg/kg body weight. The general syndrome developed as muscle tremors, increased heart rate and respiration rate followed by or concurrent with anorexia (within 24 hr) and watery, forced diarrhea (48 through 72 hr). There were signs of cardiac effects one week to ten days after administration of the drug.

Anticoccidial drugs: growth and performance depressing effects in young chickens.

Monensin, lasalocid, salinomycin, nicarbazin, halofuginone, or arprinocid were fed to 1-week-old male broiler chicks at recommended levels and 1.5, 2, 2.5, and 3 times the recommended level, for 3 weeks. Pair-feeding experiments also were conducted to investigate the extent that growth depression with medicated diets could be attributed to the drop in feed consumption. At the recommended level of drugs, growth and feed conversion were not significantly affected. At elevated drug levels, performance was impaired; the adverse effects of drugs became more pronounced with increasing the concentrations in the diets. Weight gain was significantly depressed at 1.5X with arprinocid, halofuginone, and salinomycin, at 1 to 2X with monensin, at 2X with lasalocid, and at 2.5X with nicarbazin. Feed conversion, however, was adversely affected by 2X with halofuginone or 2.5X with salinomycin, nicarbazin, arprinocid, monensin, or lasalocid. The results of the pair-feeding experiments with 2 to 3 times drug levels indicated that most of the growth depression with medicated diets could be attributed to reduced feed consumption, but all drugs except arprinocid caused some additional growth depression.

Toxic effects of lasalocid in horses.

Lasalocid was given to horses in a series of sequentially increasing single oral doses ranging between 5 and 30 mg/kg of body weight, with an appropriate washout period between treatments. One of the 5 horses died after a dosage of 15 mg/kg, 1 of 3 horses died after 21 mg/kg, 1 of 3 horses died after 22 mg/kg, and 1 of 2 horses died after 26 mg/kg. The LD50 of lasalocid for horses was estimated to be 21.5 mg/kg. Monensin was given to horses in a similar manner at dosages of 1, 2, and 3 mg/kg of body weight. One of the 2 horses died after a dosage of 2 mg/kg and 1 horse died after a dosage of 3 mg/kg. The clinical signs of toxicosis observed in horses given either drug were progressive and included depression, ataxia, paresis, and paralysis with partial anorexia. Intermittent profuse sweating was observed before death in horses given monensin.