Toxicological and transcriptomic-based analysis of monensin and sulfamethazine co-exposure on male SD rats.

Antibiotic residue has become an emerging environmental contaminant, while the toxicological effects and underlying mechanisms caused by the co-exposure to multiple veterinary antibiotics were rarely studied. In this study, male Sprague Dawley rats were exposed to monensin (M) (1, 2, 10 mg/(kg·body weight (BW)) combined with sulfamethazine (S) (60, 120, 600 mg/(kg·BW)) or single drugs for 28 consecutive days. The body weight, hematological and blood biochemical parameters, organ coefficients, and histopathology were analyzed to discover their combined toxicity effect. Transcriptomic analysis was used to reveal the possible mechanisms of their joint toxicity. Compared with the control group, the weight gain rate was significantly reduced in the H-M+S and H-S, and alkaline phosphatase in H-M+S was significantly increased. Furthermore, relative liver and kidneys weight was significantly increased, and the liver of H-M+S showed more severe lesions in histopathological analysis. For H-M+S, H-M and H-S, transcriptomic results showed that 344, 246, and 99 genes were differentially expressed, respectively. The Gene Ontology terms mainly differ in sterol biosynthetic process and steroid hydroxylase activity. The Kyoto Encyclopedia of Genes and Genome pathways showed abnormal retinol metabolism, metabolism of xenobiotics by cytochrome P450, and drug metabolism-cytochrome 450; the common 30 genes were screened from the network of protein-protein interaction. The results showed that mixed contamination of M and S produces hepatotoxicity by interfering with linoleic acid metabolism, retinol metabolism and CYP450 enzyme-dominated drug metabolism. Further analysis showed that Cyp1a2, Cyp2c61, Ugt1a3, and Ugt1a5 might be the key genes. These findings could provide more evidence for investigating the toxic effects and metabolism of mixed antibiotics contamination in mammals.

The In Vitro Cytotoxic Effects of Ionophore Exposure on Selected Cytoskeletal Proteins of C2C12 Myoblasts.

Carboxylic ionophores, such as monensin, salinomycin and lasalocid, are polyether antibiotics used widely in production animals for the control of coccidiosis, as well as for the promotion of growth and feed efficiency. Although the benefits of using ionophores are undisputed, cases of ionophore toxicosis do occur, primarily targeting the cardiac and skeletal muscles of affected animals. The 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide (MTT) viability assay was used to determine the cytotoxicity of monensin, salinomycin and lasalocid on mouse skeletal myoblasts (C2C12). Immunocytochemistry and immunofluorescent techniques were, in turn, performed to investigate the effects of the ionophores on the microfilament, microtubule and intermediate filament, i.e., desmin and synemin networks of the myoblasts. Monensin was the most cytotoxic of the three ionophores, followed by salinomycin and finally lasalocid. Monensin and salinomycin exposure resulted in the aggregation of desmin around the nuclei of affected myoblasts. The synemin, microtubule and microfilament networks were less affected; however, vesicles throughout the myoblast's cytoplasm produced gaps within the microtubule and, to a limited extent, the synemin and microfilament networks. In conclusion, ionophore exposure disrupted desmin filaments, which could contribute to the myofibrillar degeneration and necrosis seen in the skeletal muscles of animals suffering from ionophore toxicosis.

Co-exposure of Monensin Increased the Risks of Atrazine to Earthworms.

Antibiotics could enter farmlands through sewage irrigation or manure application, causing combined pollution with pesticides. Antibiotics may affect the environmental fate of pesticides and even increase their bioavailability. In this study, the influence of monensin on the degradation, toxicity, and availability of atrazine in soil-earthworm microcosms was investigated. Monensin inhibited the degradation of atrazine, changed the metabolite patterns in soil, and increased the bioavailability of atrazine in earthworms. Atrazine and monensin had a significant synergistic effect on earthworms in the acute toxic test. In long-term toxicity tests, co-exposure of atrazine and monensin also led to worse effects on earthworms including oxidative stress, energy metabolism disruption, and cocoon production compared to single exposure. The expression of tight junction proteins was down-regulated significantly by monensin, indicating that the intestinal barrier of earthworms was weakened, possibly causing the increased bioavailability of atrazine. The expressions of heat shock protein 70 (Hsp70) and reproductive and ontogenetic factors (ANN, TCTP) were all downregulated in binary exposure, indicating that the resilience and cocoon production of earthworms were further weakened under combined pollution. Monensin disturbed the energy metabolism and weakened the intestinal barrier of earthworms. These results showed that monensin increased the risks of atrazine in agricultural areas.

Acute, subacute and chronic sequelae of horses accidentally exposed to monensin-contaminated feed.

BACKGROUND: Monensin is highly toxic to horses and inadvertent ingestion can result in cardiac injury and death. OBJECTIVES: To describe sequelae of monensin ingestion and to determine clinical predictors of outcome. STUDY DESIGN: Observational clinical study. METHODS: Physical examination, electrocardiogram and echocardiography were performed on 76 horses accidentally exposed to monensin-contaminated feed. Four horses were examined within 14 days of exposure (acute period), 29 horses were examined between 15 and 45 days post-exposure (subacute period) and 70 horses were examined 4-10 months after exposure (chronic period). Follow-up information was obtained for 56 horses by telephone interviews approximately 16 months after exposure. RESULTS: Cardiac abnormalities were detected in 4/4, 19/29 and 31/70 horses during the acute, subacute and chronic periods, respectively. Sixteen months post-exposure, 34 of the 64 horses (53%) for which the outcome was known had returned to their previous use, 13 (20%) were reported to be exercise intolerant, three (5%) were retired and 14 (22%) were dead (two deaths, 12 euthanasia). Thinning of the myocardium observed at any point in time was associated with a negative outcome. Heterogeneity of the myocardium observed in the acute/subacute period was associated with a negative outcome while subjective contractile intraventricular dyssynchrony, cardiac chamber dilation, decreased fractional shortening and multiple premature ventricular complexes observed in the chronic period were associated with a negative outcome. Some horses with significant changes associated with a negative outcome in the chronic phase still returned to their previous work. MAIN LIMITATIONS: No control group and only 27 horses were examined more than once. CONCLUSIONS: Clinical outcome of horses exposed to sublethal doses of monensin is highly variable. The presence of heterogeneity and thinning of the myocardium shortly after intoxication were associated with a negative outcome.

Effect of the veterinary ionophore monensin on the structure and activity of a tropical soil bacterial community.

Monensin (MON) is a coccidiostat used as a growth promoter that can reach the environment through fertilization with manure from farm animals. To verify whether field-relevant concentrations of this drug negatively influence the structure and activity of tropical soil bacteria, plate counts, CO2 efflux measurements, phospholipid fatty acids (PLFA) and community-level physiological profiling (CLPP) profiles were obtained for soil microcosms exposed to 1 or 10 mg kg-1 of MON across 11 days. Although 53% (1 mg kg-1) to 40% (10 mg kg-1) of the MON concentrations added to the microcosms dissipated within 5 days, a subtle concentration-dependent decrease in the number of culturable bacteria (<1 log CFU g-1), reduced (-20 to -30%) or exacerbated (+25%) soil CO2 effluxes, a marked shift of non-bacterial fatty acids, and altered respiration of amines (1.22-fold decrease) and polymers (1.70-fold increase) were noted in some of the treatments. These results suggest that MON quickly killed some microorganisms and that the surviving populations were selected and metabolically stimulated. Consequently, MON should be monitored in agronomic and environmental systems as part of One Health efforts.

Environmental fate and microbial effects of monensin, lincomycin, and sulfamethazine residues in soil.

The impact of commonly-used livestock antibiotics on soil nitrogen transformations under varying redox conditions is largely unknown. Soil column incubations were conducted using three livestock antibiotics (monensin, lincomycin and sulfamethazine) to better understand the fate of the antibiotics, their effect on nitrogen transformation, and their impact on soil microbial communities under aerobic, anoxic, and denitrifying conditions. While monensin was not recovered in the effluent, lincomycin and sulfamethazine concentrations decreased slightly during transport through the columns. Sorption, and to a limited extent degradation, are likely to be the primary processes leading to antibiotic attenuation during leaching. Antibiotics also affected microbial respiration and clearly impacted nitrogen transformation. The occurrence of the three antibiotics as a mixture, as well as the occurrence of lincomycin alone affected, by inhibiting any nitrite reduction, the denitrification process. Discontinuing antibiotics additions restored microbial denitrification. Metagenomic analysis indicated that Proteobacteria, Bacteroidetes, Actinobacteria, and Chloroflexi were the predominant phyla observed throughout the study. Results suggested that episodic occurrence of antibiotics led to a temporal change in microbial community composition in the upper portion of the columns while only transient changes occurred in the lower portion. Thus, the occurrence of high concentrations of veterinary antibiotic residues could impact nitrogen cycling in soils receiving wastewater runoff or manure applications with potential longer-term microbial community changes possible at higher antibiotic concentrations.

Hazard assessment of the veterinary pharmaceuticals monensin and nicarbazin using a soil test battery.

Veterinary pharmaceuticals are widely used as food additives in the poultry industry, and the unknown consequences of releasing these compounds into the environment are of concern. The purpose of the present study was to determine the direct impact of 2 veterinary pharmaceuticals (nicarbazin and monensin), commonly used in the poultry industry, on nontarget invertebrates and plant species. Ecotoxicological tests were used to evaluate the acute and chronic toxicity in earthworms (Eisenia andrei), collembolans (Folsomia candida), and 2 plant species (Brassica rapa and Triticum aestivum). Chemical analytical measurements were in good agreement with the nominal concentrations used, although some variability was seen. The results obtained showed no effects of nicarbazin at the highest nominal tested concentration of 1000 mg a.i./kg soil dry weight on any of the organisms, whereas exposure to monensin caused a concentration-specific response pattern. Species sensitivity to monensin decreased in the following rank order: B. rapa > T. aestivum > E. andrei > F. candida, with measured median effect concentrations (based on soil exposure) ranging between approximately 10 and 120 mg/kg. Our results emphasize the importance of using a test battery when assessing ecotoxicological effects by using different ecophysiological endpoints and species from different trophic levels. Environ Toxicol Chem 2018;37:3145-3153. © 2018 SETAC.

Impact of ionophore monensin on performance and Cu uptake in earthworm Eisenia andrei exposed to copper-contaminated soil.

Exposure of beneficial soil organisms to chemical mixtures is of great concern and can result in unexpected deleterious consequences. We investigated the effects of concurrent soil contamination with monensin, a veterinary pharmaceutical and feed additive, and copper, on earthworm copper uptake and reproductive success. The animals were exposed for 14 or 28 days to both substances and the results showed that the Cu body burden of earthworms increases in the presence of monensin. The harmful effects of Cu on earthworm cocoon production were considerably higher when monensin was also present in the soil. To localise the copper in earthworm tissues, histological staining was performed using two different dyes (rubeanic acid and 5-4-(p-dimethylaminobenzylidene)-rhodanine). Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to quantify the Cu levels in the tissues. Cu was found predominantly in the gut wall. The Cu content in the body wall was at least ten times lower compared to the gut, but was proportional to the level of soil contamination. Concurrent soil contamination with monensin and copper resulted in higher earthworm Cu levels and in decreased reproductive success of these important soil decomposers.

Cytotoxicity of monensin, narasin and salinomycin and their interaction with silybin in HepG2, LMH and L6 cell cultures.

The cytotoxic effect of monensin, narasin and salinomycin followed by their co-action with silybin in the cell line cultures of human hepatoma (HepG2), chicken hepatoma (LMH) or rat myoblasts (L6) have been investigated. The effective concentration of the studied ionophoric polyethers has been assessed within two biochemical endpoints: mitochondrial activity (MTT assay) and membrane integrity (LDH assay) after 24h incubation of each compound and farther, the cytotoxicity influenced in course of their interaction with silybin was determined. The most affected endpoints were found for inhibition of mitochondrial activity of the hepatoma cell lines and their viability depended on concentration of the ionophoric polyether, as well as on the cell line tested. The rat myoblasts were more sensitive target for cellular membrane damage when compared to inhibition of mitochondrial activity. An interaction between the ionophoric polyethers and silybin resulted a considerable cytotoxicity decrease within all studied cell lines; the combination index (CI) showed differences of interaction mode and dependence on cell culture, concentration of silybin, as well as the assay used. The obtained results are of interest in respect to recent findings on applicability of salinomycin and monensin for human therapy.

Antimicrobial potential of the ionophore monensin on freshwater biofilm bacteria.

Microorganisms play key roles in stream ecosystems, but comparatively little is known about the resilience of freshwater bacterial communities and their susceptibility to the chemical by-products of agricultural land use. Antibiotics used in the agricultural sector are of particular concern and have been detected in waterways associated with agricultural land. Despite widespread agricultural intensification globally and the sector's high antibiotic use, the effects of agricultural antibiotic by-products on stream microbial communities have yet to be characterised. We investigated the impacts of the antibiotic monensin on microbial biofilm communities in a simulated contamination event using streamside-replicated channels. A 24-h pulse experiment in flow channels precolonised by stream biofilm microbial communities contrasted the effects of monensin concentrations ranging from realistic to extreme toxicity levels (1-550 ug L(-1)). Biofilm community composition was characterised immediately before and after the pulse for several weeks using automated ribosomal intergenic spacer analysis. Despite applying acutely toxic levels of monensin, only limited effects to biofilm community composition were detected immediately after antibiotic application, and these disappeared within 4 days. Rather, temporal factors drove biofilm differences, highlighting the overriding importance of wider, catchment-level, physiochemical hydrological influences on structuring freshwater biofilm communities, as opposed to localised and sporadic agricultural surface runoff contamination events containing antibiotics.

Testicular effects of monensin, a golgi interfering agent in male rats.

OBJECTIVE: The present study was undertaken to explore the effects of monensin, a potent Golgi disturbing agent on male fertility. METHODS: Male Wistar rats were administered monensin at the dose levels of 2.5, 5, and 10 mg/kg b wt. Animals were sacrificed after 67 days of the treatment. The activities of lactate dehydrogenase (LDH), ATPase, acid phosphatase and thiamine pyrophosphatase (TPPase) were measured in the testis. Cytochemical assay of Golgi body marker enzyme, thiamine pyrophosphatase was also performed. Ultrastructural changes in testis were studied by Transmission electron microscopy. Sperm number and motility were also examined. RESULTS AND DISCUSSION: The alterations in the activities of above mentioned enzymes indicate the pronounced effect of the drug on the functioning of spermatogenic cells. The findings from electron microscopy such as membrane disruption, swelling and disintegration of Golgi apparatus strongly suggest the interference of monensin with the functioning of Golgi apparatus in the spermatogenic cells. Data from the sperm number and motility as well as the fertility studies and the resulted litter size further points towards the antifertility effects of monensin in male rats. CONCLUSION: The findings from the present study strongly indicated the effects of monensin on the testis, involving alterations in key enzyme activities and changes at the ultrastructural level.

Acute monensin toxicosis in broiler breeder chickens.

Peracute onset of disease was reported in a 42-wk-old broiler breeder flock that was presented by error with feed containing monensin at approximately seven times the approved level for broiler chickens. Morbidity and mortality were extremely high, and the affected chickens displayed feed refusal, decreased water consumption, and severe paralysis that ranged from abnormal gait to a complete inability to move. During the first 10 days postingestion of the suspect feed, mortality in hens reached 13.7% and 70.9% in the roosters. Hen day production decreased from 67% to 3% in the same period of time. A total of 638 g/ton of monensin was detected in suspect feed samples by one laboratory and 740 g/ton in a second laboratory. Twenty-one days after removal of the suspect feed, the mortality rate returned to normal levels in both hens and roosters, albeit feed consumption and egg production remained extremely low, which prompted the company involved to eliminate the flock.

Does monensin in chicken manure from poultry farms pose a threat to soil invertebrates?

Monensin is a carboxylic polyether ionophore used in the poultry industry as a coccidiostat. It enters the environment via manure from broiler farms. In spite of its potential presence in the environment, information concerning monensin residues in manure and soil and its toxicity to soil organisms are insufficient. In the present study, two beneficial soil invertebrate species, earthworms (Eisenia andrei) and woodlice (Porcellio scaber), were used to assess the toxicity of monensin. Animals were exposed to a range of monensin concentrations via soil or food. Earthworm reproduction was found to be the most susceptible endpoint (NOEC=3.5 mg kg(-1) dry soil; EC(50)=12.7 mg kg(-1) dry soil), while no adverse effects were recorded in isopods (NOEC⩾849mgkg(-1) dry soil, NOEC⩾357mgkg(-1) dry food). The obtained toxicity data were compared with potential concentrations of monensin in soil. In view of this, manure from broiler chickens treated with monensin at a poultry farm was sampled. According to monensin and nitrogen concentrations in the chicken manure and the degradation time of monensin, the predicted environmental concentration (PEC) was calculated. PEC of monensin is around 0.013 mg kg(-1) soil if manure is used after 3 months of composting and 0.05 mg kg(-1) soil if used without storage. Data for earthworm reproduction was used to estimate the predicted no-effect concentration (PNEC). If fresh chicken manure is applied to terrestrial ecosystems, the risk quotient (PEC/PNEC ratio) is above 1, which indicates that monensin might pose an environmental risk under certain conditions. To prevent this, it is strongly recommended to compost chicken manure for several months before using it as fertiliser.

Measurement of plasma cardiac troponin I concentration by use of a point-of-care analyzer in clinically normal horses and horses with experimentally induced cardiac disease.

OBJECTIVE: To compare cardiac troponin I (cTnI) concentrations determined by use of a point-of-care analyzer with values determined by use of a bench-top immunoassay in plasma samples obtained from clinically normal horses with and without experimentally induced cardiac disease, and to establish a reference range for plasma equine cTnI concentration determined by use of the point-of-care analyzer. ANIMALS: 83 clinically normal horses, 6 of which were administered monensin to induce cardiac disease. PROCEDURES: A blood sample was collected from each of the 83 clinically normal horses to provide plasma for analysis by use of the point-of-care analyzer; some of the same samples were also analyzed by use of the immunoassay. All 83 samples were used to establish an analyzer-specific reference range for plasma cTnI concentration in clinically normal horses. In 6 horses, blood samples were also collected at various time points after administration of a single dose of monensin (1.0 to 1.5 mg/kg) via nasogastric intubation; plasma cTnI concentration in those samples was assessed by use of both methods. RESULTS: The analyzer-specific reference range for plasma cTnI concentration in clinically normal horses was 0.0 to 0.06 ng/mL. Following monensin treatment in 5 horses, increases in plasma cTnI concentration determined by use of the 2 methods were highly correlated (Pearson correlation, 0.83). Peak analyzer-determined plasma cTnI concentrations in monensin-treated horses ranged from 0.08 to 3.68 ng/mL. CONCLUSIONS AND CLINICAL RELEVANCE: In horses with and without experimentally induced cardiac disease, the point-of-care analyzer and bench-top immunoassay provided similar values of plasma cTnI concentration.

Clinical findings and serum cardiac troponin I concentrations in horses after intragastric administration of sodium monensin.

Six adult horses were administered sodium monensin, 1.0-1.5 mg/kg, via gastric gavage. Anorexia and/or diarrhea occurred within 24 hr after monensin administration in all 6 horses. Cardiac disease and dysfunction were evaluated by both elevations in heart rate, echocardiography, and an increase in serum concentrations of cardiac troponin I (cTnI), occurred in 4 horses. The development and severity of cardiac disease was likely affected by the monensin dose, vehicle (water or corn oil) mixed with monensin, and/or whether the monensin was administered to fed or fasted horses. Initial increases in cTnI concentrations occurred between 24 and 72 hr after monensin administration. The 2 horses with the highest cTnI concentrations died or were euthanized within 5 days after monensin administration and had severe cardiac disease. One horse had increased cTnI concentrations from day 2 to day 16, but no apparent change in ventricular contractile function was evident on echocardiography. The fourth diseased horse did not return to cTnI reference intervals until day 27 after monensin administration, and the ventricular function was still abnormal just before euthanasia 9 months later. Cardiac troponin I measurements could be useful in managing farm outbreaks of accidental monensin feeding by the early identification of horses with cardiac disease.

Protective effects of IRFI-042 in monensin induced neurotoxicity in chicks.

Monensin, a well known ionophore antibiotic, may cause severe damage in neuronal cells by altering Na+/K+-ATPase and Ca2+-ATPase. We investigated whether IRFI-042, a synthetic analogue of vitamin E, may block lipid peroxidation in neuronal cells and protect against monensin neurotoxicity in chicks. Monensin toxicity was induced in chicks by once-daily administration (150 mg/kg by oral gavages), for 8 days. Sham animals received a saline solution and were used as controls. All animals were randomized to receive either IRFI-042 (20 mg/kg) or its vehicle. Survival rate, brain lipid peroxidation, mRNA for neuronal and inducible nitric oxide synthases (nNOS and iNOS) and brain histological evaluations, including immunohistochemical expression of nNOS and iNOS were performed. Monensin administration decreased survival rate, induced behavioural changes, increased brain lipid peroxidation, reduced brain nNOS mRNA and immunostaining and enhanced iNOS mRNA and immunostaining in the brain in chicks. IRFI-042 significantly improved the survival rate and counteracted monensin-induced changes in chick brains. Our data suggest that monensin is responsible of neurotoxicity in chicks by inducing oxidative stress/lipid peroxidation and that IRFI-042 might represent a useful pharmacological approach to protect against the neuronal damage induced by this monovalent carboxylic ionophorous polyether antibiotic.

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.

Monensin is not toxic to aquatic macrophytes at environmentally relevant concentrations.

Monensin, a common livestock feed additive, has been detected in surface waters around areas of intensive agriculture. The effect of this ionophore antibiotic on floating (Lemna gibba) and submersed (Myriophyllum spicatum, Elodea canadensis, Egeria densa) freshwater macrophytes was investigated under seminatural field conditions using 12,000 l of outdoor microcosms. Exposure concentrations of 0, 12, 25, 50, and 100 mug/l (n = 3) were evaluated over a 35-day period. Submersed plants were grown individually in 115-ml plastic "cone-tainers" and assessed for various growth and pigment end points. E. canadensis and M. spicatum also were grown in assemblages to represent model populations and two-species communities. Few statistically significant differences from control organisms were observed for any of the monitored end points. Overall, monensin is deemed unlikely to cause toxicity in freshwater macrophytes at current environmental concentrations. However, the ability to characterize toxicity in macrophytes is based partially on the relative growth rates (RGRs) of the plants. The greater the RGR, the more sensitive the assay may be to contaminants. The RGRs of E. canadensis and M. spicatum grown in model populations and communities were found to be significantly higher than the RGRs of plants grown individually. This implies that the "cone-tainer" method, although simple and easy to perform, may underestimate toxicity in simulated field studies.