Integrated comparison of growth and oxidative stress induced by tylosin in two freshwater algae Chlorella vulgaris and Raphidocelis subcapitata.

Two model algae, Chlorella vulgaris (C. vulgaris) and Raphidocelis subcapitata (R. subcapitata), are commonly used in registration procedures to evaluate compounds with antimicrobial capacity. However, it has been found that these two algae show considerable differences in sensitivity when exposed to antibiotics. The selection of a suitable test species plays a crucial role in assessing the environmental hazards and risks of a compound, as the balance between oxidative stress and antioxidants is a key factor for alga growth. This study was conducted to investigate the status of oxidative stress and mechanism of antioxidant defense system of algae under antibiotic stress. Different tylosin (TYN) exposure-concentrations were used for the tests in this study. Oxidative stress biomarkers (malondialdehyde (MDA)), non-enzymatic antioxidants (reduced glutathione (GSH)), antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GP), glutathione S-transferase (GST)) and photosynthetic pigments were measured to determine the status of the antioxidant defense system. With increasing TYN concentration, the growth of R. subcapitata was significantly inhibited, while there was no effect on C. vulgaris. When the growth of R. subcapitata was inhibited, the content of MDA was significantly increased and the antioxidant system was activated, which indicated a significant increase in the activity of SOD and CAT.

Preparation and evaluation of tilmicosin-loaded hydrogenated castor oil nanoparticle suspensions of different particle sizes.

Three tilmicosin-loaded hydrogenated castor oil nanoparticle (TMS-HCO-NP) suspensions of different particle sizes were prepared with different polyvinyl alcohol surfactant concentrations using a hot homogenization and ultrasonic technique. The in vitro release, in vitro antibacterial activity, mammalian cytotoxicity, acute toxicity in mice, and stability study were conducted to evaluate the characteristics of the suspensions. The in vitro tilmicosin release rate, antibacterial activity, mammalian cytotoxicity, acute toxicity in mice, and stability of the suspensions were evaluated. When prepared with polyvinyl alcohol concentrations of 0.2%, 1%, and 5%, the mean diameters of the nanoparticles in the three suspensions were 920±35 nm, 452±10 nm, and 151±4 nm, respectively. The three suspensions displayed biphasic release profiles similar to that of freeze-dried TMS-HCO-NP powders, with the exception of having a faster initial release. Moreover, suspensions of smaller-sized particles showed faster initial release, and lower minimum inhibitory concentrations and minimum bactericidal concentrations. Time-kill curves showed that within 12 hours, the suspension with the 151 nm particles had the most potent bactericidal activity, but later, the suspensions with larger-sized particles showed increased antibacterial activity. None of the three suspensions were cytotoxic at clinical dosage levels. At higher drug concentrations, all three suspensions showed similar concentration-dependent cytotoxicity. The suspension with the smallest-sized particle showed significantly more acute toxicity in mice, perhaps due to faster drug release. All three suspensions exhibited good stability at 4°C and at room temperature for at least 6 months. These results demonstrate that TMS-HCO-NP suspensions can be a promising formulation for tilmicosin, and that nanoparticle size can be an important consideration for formulation development.

Acute toxicity study of tilmicosin-loaded hydrogenated castor oil-solid lipid nanoparticles.

BACKGROUND: Our previous studies demonstrated that tilmicosin-loaded hydrogenated castor oil solid lipid nanoparticles (Til-HCO-SLN) are a promising formulation for enhanced pharmacological activity and therapeutic efficacy in veterinary use. The purpose of this work was to evaluate the acute toxicity of Til-HCO-SLN. METHODS: Two nanoparticle doses were used for the study in ICR mice. The low dose (766 mg/kg.bw) with tilmicosin 7.5 times of the clinic dosage and below the median lethal dose (LD(50)) was subcutaneously administered twice on the first and 7th day. The single high dose (5 g/kg.bw) was the practical upper limit in an acute toxicity study and was administered subcutaneously on the first day. Blank HCO-SLN, native tilmicosin, and saline solution were included as controls. After medication, animals were monitored over 14 days, and then necropsied. Signs of toxicity were evaluated via mortality, symptoms of treatment effect, gross and microscopic pathology, and hematologic and biochemical parameters. RESULTS: After administration of native tilmicosin, all mice died within 2 h in the high dose group, in the low dose group 3 died after the first and 2 died after the second injections. The surviving mice in the tilmicosin low dose group showed hypoactivity, accelerated breath, gloomy spirit and lethargy. In contrast, all mice in Til-HCO-SLN and blank HCO-SLN groups survived at both low and high doses. The high nanoparticle dose induced transient clinical symptoms of treatment effect such as transient reversible action retardation, anorexy and gloomy spirit, increased spleen and liver coefficients and decreased heart coefficients, microscopic pathological changes of liver, spleen and heart, and minor changes in hematologic and biochemical parameters, but no adverse effects were observed in the nanoparticle low dose group. CONCLUSIONS: The results revealed that the LD50 of Til-HCO-SLN and blank HCO-SLN exceeded 5 g/kg.bw and thus the nanoparticles are considered low toxic according to the toxicity categories of chemicals. Moreover, HCO-SLN significantly decreased the toxicity of tilmicosin. Normal clinic dosage of Til-HCO-SLN is safe as evaluated by acute toxicity.

Hydrogenated castor oil nanoparticles as carriers for the subcutaneous administration of tilmicosin: in vitro and in vivo studies.

Tilmicosin-loaded solid lipid nanoparticles (SLN) were prepared with hydrogenated castor oil (HCO) by o/w emulsion-solvent evaporation technique. The nanoparticle diameters, surface charges, drug loadings and encapsulation efficiencies of different formulations were 90 approximately 230 nm, -6.5 approximately -12.5 mV, 40.3 approximately 59.2% and 5.7 approximately 11.7% (w/w), respectively. In vitro release studies of the tilmicosin-loaded nanoparticles showed a sustained release and the released tilmicosin had the same antibacterial activity as that of the free drug. Pharmacokinetics study after subcutaneous administration to Balb/c mice demonstrated that a single dose of tilmicosin-loaded nanoparticles resulted in sustained serum drug levels (>0.1 microg/mL) for 8 days, as compared with only 5 h for the same amount of tilmicosin phosphate solution. The time to maximum concentration (Tmax), half-life of absorption (T(1/2) ab) and half-life of elimination (T(1/2) el) of tilmicosin-loaded nanoparticles were much longer than those of tilmicosin phosphate solution. Tissue section showed that drug-loaded nanoparticles caused no inflammation at the injection site. Cytotoxicity study in cell culture and acute toxicity test in mice demonstrated that the nanoparticles had little or no toxicity. The results of this exploratory study suggest that the HCO-SLN could be a useful system for the delivery of tilmicosin by subcutaneous administration.

The possible protective effect of L-carnitine on tilmicosin-induced cardiotoxicity in mice.

The protective effect of L-carnitine was investigated against tilmicosin-induced cardiotoxic effects including blood creatine kinase (CK), CK-MB, total sialic acid as well as the alterations in glutathione and malondialdehyde concentrations in mice. Thirty-two Balb/C mice were divided into four groups including group 1 (control), group 2 (L-carnitine, s.c., 500 mg/kg for 5 days), group 3 (tilmicosin, s.c., single dose of 75 mg/kg) and group 4 (L-carnitine plus tilmicosin). Serum CK, CK-MB and malondialdehyde (MDA) levels were significantly (P < 0.05) higher in group 3 compared with those of other groups. Total sialic acid level in group 3 was found to be significantly (P < 0.05) higher than that in groups 1 and 2, as well. Contrary to these results, glutathione level in group 3 was found to be significantly (P < 0.05) lower than that in groups 1 and 2. In group 4, serum CK, CK-MB, MDA and total sialic acid levels were found to be significantly (P < 0.05) lower than those in group 3. These results suggest that tilmicosin is cardiotoxic in mice as evidenced by higher total sialic acid, CK and CK-MB. In addition, tilmicosin caused the decrease in glutathione and increase in MDA levels. However, administration of L-carnitine could ameliorate these adverse toxic effects of tilmicosin in mice.

Tylosin does not affect GnRH-induced LH secretion in rams.

The aim of this study was to investigate the effect of a therapeutic dose of Tylosin (Tylan 50) on gonadotropin releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion in sheep. A total of 10 mature rams were divided into two groups by balancing body weights (bw) and body condition scores. Five of the rams received 10mg/kg Tylosin intramuscularly (i.m., Tylosin group), while the other five were given placebo (Control group), for 5 days. On Day 5, all the rams were injected intravenously (i.v.) with the GnRH agonist Ovarelin at 0.1 microg/kg bw. Blood samples were collected at -30, 0, 30, 60, 90, 120, 150, 180, 210, 240, and 270 min for measuring LH levels in the plasma. Three days after the cessation of Tylosin application (Day 8) the injection of GnRH was repeated at the same dose. Although LH secretion appeared to be lower on Day 8 compared to Day 5, there were no significant differences between the groups for the mean LH concentrations, total LH secretion, peak LH concentrations, timing of LH peak, duration of LH secretion, and LH secretions on Days 5 and 8. These results indicate the absence of a negative effect of a therapeutic dose of Tylosin on GnRH-induced LH secretion in rams.