Bisphenol A in edible tissues of rams exposed to repeated low-level dietary dose by high-performance liquid chromatography with fluorescence detection.

The presented work deals with levels and distribution of bisphenol A (BPA) in the edible tissues of a large food-producing animal species. An experimental animal study included 14 young Istrian pramenka rams (Ovis aries), of which seven were exposed for 64 days to a low dietary dose of BPA at 25 µg/kg b.w./day, and seven served as a control group. Residue analysis of both aglycone and total BPA was performed in the muscle tissue, liver, kidney and fat tissue of the individual animals by means of enzymatic deconjugation (for total BPA), organic solvent extraction, molecularly imprinted polymer solid-phase extraction (MISPE) clean-up and high-performance liquid chromatography with fluorescence detection (HPLC-FLU). The analysis was optimized and validated for aglycone BPA in the fat tissue and for the total BPA in all tissues investigated. Edible tissues of the control group of rams generally remained BPA-free, while there were concentration differences between the control and treated groups for liver and kidney post last administration. The human health risk resulting from this study was assessed by the estimated dietary exposure in adults, which was < 0.1% related to the valid European Union Tolerable Daily Intake (TDI) value of 4 µg/kg b.w./day. However, it would be 58-fold higher than the newly proposed TDI value of 0.04 ng/kg b.w./day.

Basic Exploratory Study of Bisphenol A (BPA) Dietary Administration to Istrian Pramenka Rams and Male Toxicity Investigation.

Bisphenol A (BPA), an endocrine-disrupting chemical and environmental pollutant, has been reported by many researchers to induce male reproductive toxicity in different experimental models. In this study, we investigated whether long-term exposure for two months to 25 µg/kg body weight (low dose) of BPA affects spermatogenesis or sperm quality in young Istrian Pramenka rams exposed via diet. We evaluated body and testicular weights, histopathology of testes and epididymides, and sperm analyses, and compared these parameters between the group of treated rams and the control group of rams. Although there were some differences between the two groups, these differences were not large or statistically significant. The only statistically significant difference was the lower epithelial height of seminiferous tubules in treated rams, compared to control rams. In addition to assessing toxicity, BPA concentrations in the blood plasma of treated rams were determined after the first administration, and the toxicokinetic parameters of total BPA were calculated. In this study, no major signs of altered reproduction in rams were detected.

Determination of free and total bisphenol A in the urine and feces of orally and subcutaneously dosed sheep by high-performance liquid chromatography with fluorescence detection.

An analytical procedure has been introduced to enable a study of the excretion of free bisphenol A (BPA), total BPA and its main metabolite bisphenol A glucuronide (BPA-GLUC). In the experiment, in which 100 µg/kg b. w. BPA was administered daily to one Istrian Pramenka sheep for 5 days with consecutive urine and feces samples being taken, BPA and total BPA were determined in samples using high-performance liquid chromatography (HPLC) with fluorescence detection. Because of their good recovery, precision, and sensitivity, the methods have also proved applicable to further ecotoxicological studies of free BPA, BPA-GLUC and total BPA. The results were subsequently compared with reported field studies of BPA in livestock excreta.

Preliminary toxicokinetic study of BPA in lactating dairy sheep after repeated dietary and subcutaneous administration.

Dietary intake is the predominant route of human exposure to bisphenol A and one of the important food commodities is milk. The aim of our study was to preliminarily evaluate the bisphenol A exposure and disposition in sheep milk after repeated dietary and subcutaneous administration of a relatively low dose (100 µg/kg of b. w./day) of bisphenol A to a sheep. On the basis of blood plasma sampling, milk sampling and HPLC analysis, we developed the toxicokinetic model. With the toxicokinetic model we showed that most likely only free bisphenol A passes into the mammary gland and is subsequently conjugated there. The percentage of the dose eliminated with milk was less than 0.1%, regardless of the route of bisphenol A administration. It is proven that the bisphenol A is eliminated through the milk of lactating sheep. However, the amounts excreted in the milk that were detected in this study are minimal.

Uterine heterologous malignant mixed Müllerian tumor in a dwarf rabbit (Oryctolagus cuniculus).

Malignant mixed Müllerian tumor (MMMT) is a rare neoplasm of the female genital system. A case of MMMT in the uterus of an 8-year-old female dwarf rabbit, which died with clinical signs associated with severe acute dyspnea and anorexia, is described. At necropsy, an oval, firm tumor was found in each of the 2 uterine horns. Numerous metastases were scattered throughout the mediastinum, thoracic diaphragm, and all pulmonary lobes. Microscopically, the tumors consisted of a poorly demarcated, unencapsulated neoplasm, composed of closely associated carcinomatous and sarcomatous components and areas of osteosarcomatous differentiation. Metastases were composed entirely of the sarcomatous component with osteosarcomatous differentiation. Immunohistochemically, the neoplastic epithelial component was positive for cytokeratin and negative for α-smooth muscle actin (α-SMA), vimentin, and desmin. The sarcomatous component was diffusely and strongly positive for vimentin, focally positive for α-SMA (<20% of cells positive), and negative for desmin. The neoplasm was diagnosed as a heterologous MMMT with metastases to the lung, mediastinum, and thoracic diaphragm.

Influence of P-glycoprotein inhibition on secretion of ivermectin and doramectin by milk in lactating sheep.

The aim of to the present study was to evaluate the effects of verapamil (VER) on plasma pharmacokinetics of ivermectin (IVM) and doramectin (DOR) in lactating Istrian Pramenka dairy sheep and to investigate the role of P-glycoprotein (P-gp) in transport of avermectins into milk. Pharmacokinetics of IVM and DOR following subcutaneous administration of 0.2mg/kg b.w. was evaluated in four groups of sheep. They were administered either IVM or DOR alone or in combination with verapamil (VER) at a dose of 3.0mg/kg b.w., 3 times at 12h intervals. Blood plasma and milk samples were collected at defined time intervals over 30 days post-treatment to determine IVM and DOR concentration levels. Pharmacokinetic parameters in sheep injected with IVM or DOR alone corresponded to previously published values. Comparison between sheep injected with IVM only, and sheep injected with IVM in combination with VER (IVM+VER) showed significant difference in pharmacokinetic parameters in blood plasma. Area under the concentration-time curve (AUC) truncated at 2 days (AUC(2)) was 15 and 28 µg day/L for group IVM and IVM+VER, respectively. With co-administration of VER, apparent plasma clearance (Cl/F) and mean residence time (MRT) of IVM decreased from 135 to 116 L/day and from 5.8 to 3.8 days, respectively. Similar trends were observed for DOR (AUC(2) 48 vs. 68 µg day/L, Cl/F 61 vs. 46 L/day, and MRT 5.6 vs. 4.4 days for groups DOR and DOR+VER, respectively). This study confirms that co-administration of VER has a significant effect on pharmacokinetic parameters of subcutaneously administered IVM in blood plasma. The influence on DOR pharmacokinetics is much weaker. This could be either due to the difference in lipophilicity or the difference in affinity towards P-gp as a result of structural differences. No significant influence of VER on AUC ratio of IVM and DOR between milk and plasma was observed suggesting that P-gp does not govern transport of avermectins into milk.

Factors influencing dissipation of avermectins in sheep faeces.

Factors influencing fate of avermectins (abamectin, doramectin) in faeces of treated sheep were investigated under different experimental conditions. In the laboratory, concentrations of both avermectins were declined in homogenised faeces of treated animals until day 14 of exposure, regardless of experimental conditions. After that day, no significant decrease in concentrations was observed till the end of the experiment. Established DT(50) did not exceed 9 days. In the karst pasture, an average DT(50) of 27 days was established for abamectin and 23 days for doramectin in natural faeces of treated sheep. In the compost mixture, doramectin concentration was decreased by 38.9+/-2.6% during 21 days of the thermophilic phase of composting. Therefore, DT(50) was not established. A possible influence of moisture content of sheep faeces on concentrations of avermectins was observed.

Linearity of eprinomectin pharmacokinetics in lactating dairy sheep following pour-on administration: excretion in milk and exposure of suckling lambs.

Pharmacokinetics of eprinomectin (EPR) were studied in blood plasma and milk in two groups of six Istrian Pramenka dairy sheep and their suckling lambs following pour-on administration of EPR to ewes at dose levels of 0.5 and 1mg/kg. Maximum concentration in plasma was 2.22 and 5.25 microg/l, and AUC was 13.6 and 33.7 microg day/l for the 0.5 and 1.0mg/kg dose, respectively. These results indicate that drug exposure with a dose of 0.5mg/kg, which is commonly used in cattle, may be subtherapeutic. The concentration time course in milk paralleled plasma concentrations. In the dose range studied, linear pharmacokinetics of EPR were demonstrated. Milk-to-plasma AUC ratio was 0.79+/-0.12 and 1.12+/-0.43; the fraction of dose recovered in milk was 0.037+/-0.011 and 0.058+/-0.027% for the low and high dose, respectively. Maximum residual levels in milk were below the maximum acceptable level of 20 microg/kg; however, EPR was detected in all samples investigated. Despite low permeability in milk, AUC in plasma of suckling lambs was between 20 and 30% of the AUC in plasma of ewes.

Kinetics of abamectin disposition in blood plasma and milk of lactating dairy sheep and suckling lambs.

Abamectin (ABM) has been used worldwide as an anthelmintic drug in veterinary medicine and as an agricultural pesticide. Its pharmacokinetics and permeation into milk was evaluated in dairy sheep after subcutaneous administration. ABM elimination half-lives and mean residence times were 1.7 and 3.7 days for blood plasma and 1.9 and 3.8 days for milk, respectively. The ABM milk to plasma concentration ratio (0.89) primarily depends on milk fat content. Transfer of ABM residues to suckling lambs was evaluated by determination of ABM concentration time courses in lambs' plasma. Mean maximal concentration in lambs was 1.6 microg L(-1) at 3.3 days, and elimination half-life was 2.7 days. In ewes' plasma and milk, ABM was detected up to 23 days. Because of different pharmacokinetics, ABM exposure in lambs was almost 10% of the exposure in ewes, although the amount excreted in milk was only 1.0% of the dose.

Time profile of abamectin and doramectin excretion and degradation in sheep faeces.

We studied abamectin and doramectin excretion and their degradation in sheep faeces under field conditions on pasture after a single subcutaneous dose (0.2mg/kg body weight). In the excretion experiment, maximal abamectin concentration (1277 ng/g dry faeces) was detected on day 3, while doramectin concentration showed two peaks (2186 and 1780 ng/g dry faeces on days 2 and 5, respectively). Both avermectins were excreted at approximately the same rate (k=0.23 day(-1) for abamectin and 0.19 day(-1) for doramectin). In the field, a rapid loss of abamectin and doramectin from sheep faeces was seen during the first 32 days after which concentrations remained constant at approximately 77 ng/g and 300 ng/g, respectively. The half life values (DT(50)) for abamectin and doramectin dissipation from sheep faeces were 23 and 22 days, respectively, during the first 32 days. Dissipation of both avermectins was strongly correlated with moisture content of the faeces.

Degradation of abamectin and doramectin on sheep grazed pasture.

Avermectins are widely used veterinary medicines. They bind strongly to faeces in their non-metabolized form and their half-life in faeces depends on field conditions. There are conflicting data regarding the behaviour of avermectins in the environment. Therefore, we investigated the degradation of abamectin and doramectin on sheep grazed pasture under field conditions in soil, soil-faeces and faeces samples from day 6 to day 70 (abamectin) or to day 50 (doramectin) after sheep treatment. Field conditions were recorded periodically during the experiment. Degradation of abamectin in sheep faeces and in soil-faeces was observed until day 60, with small amounts present in faeces until 70 days post treatment. Because the concentration of abamectin residues in soil was very low on day 6 after treatment, further significant degradation could not be measured. The concentration of doramectin in all analysed matrices decreased rapidly until day 50. It can be concluded that high concentrations of both avermectins were present during the first 20 days after treatment and that field conditions have an important role in degradation of avermectins on grazed pasture of treated animals. Clear identification of the consequences of avermectin exposure and the period of the greatest environmental risk will require further investigations.

Subchronic liver injuries caused by microcystins.

The subchronic effects of cyanobacterial lyophilizate (CL) containing microcystins on liver were investigated in female New Zealand rabbits. Sterilised CL containing microcystins was injected i.p. Liver toxicity was assessed by histological examination of liver samples. Non-invasive magnetic resonance imaging (MRI) of liver was also performed in order to assess changes in the homogeneity of liver tissue. Subchronical intoxication with microcystins caused morphological changes of liver tissue that were also detected by use of MRI. Histological analysis showed that changes seen on MRI represent liver injury characterised with fatty infiltration and periportal fibrosis. This demonstrates that subchronic exposure to microcystins can lead to liver degeneration, which can easily be detected in vivo by use of MRI.