P. Badino

Effects of clenbuterol as a repartitioning agent onβ-Adrenoceptor concentrations in heart, bronchi and brain of veal calves

The effects induced by dietary clenbuterol (20 micrograms kg-1 body weight day-1 for 40 days) on beta-adrenergic receptor (beta-AR) subtypes in the heart, bronchial smooth muscles and the CNS of veal calves were investigated using a binding method. Clenbuterol exposure caused a significant (P < 0.05, P < 0.01, P < 0.001) decrease in beta 1-AR and beta 2-AR in both cardiac atria and ventricles of treated animals (excluding the beta 2-AR of the right atrium). Similarly, a significant (P < 0.01, P < 0.001) down-regulation of beta-AR subtypes in bronchial smooth muscles of treated calves was observed. In the CNS (cerebral cortex, hippocampus, hypothalamus and cerebellum) the down-regulation was limited to beta 2-AR, with the exception of the hippocampus in which both beta 1-AR and beta 2-AR concentrations were significantly (P < 0.05; P < 0.01) reduced. Scatchard analysis of the binding of the beta-AR antagonist, (-) [3H]CGP 12177, revealed that the down-regulation of beta-AR was not associated with any modification in binding affinity, as Kd values were unaffected by clenbuterol treatment. Data obtained indicated that prolonged clenbuterol exposure induced a remarkable beta-AR down-regulation in the heart, bronchi and brain of veal calves.

A study to compare circulating flunixin, meloxicam and gabapentin concentrations with prostaglandin E2 levels in calves undergoing dehorning

The purpose of this study was to investigate the pharmacokinetics of intravenous flunixin (2.2 mg/kg b.w.), oral meloxicam (1mg/kg b.w.), oral gabapentin (15 mg/kg b.w.) alone or co-administrated with meloxicam as well as the effects of these compounds on prostaglandin E2 (PGE2) synthesis in calves subjected to surgical dehorning. Plasma samples collected up to 24h after drug administration were analyzed by liquid chromatography/mass spectrometry, whereas blood PGE2 levels were measured by immunoenzymatic assay. In plasma, the terminal half-live of flunixin, meloxicam and gabapentin were 6.0 h (range, 3.4-11.0 h), 16.7h (range, 13.7-21.3h) and 15.3h (range, 11-32.9h), respectively. The co-administration of single doses of gabapentin and meloxicam did not seem to affect the pharmacokinetic profile of the two drugs except for gabapentin that reached significantly (P<0.05) higher maximum serum concentration (Cmax) when co-administered with meloxicam, than when administered alone. At 5, 360 and 720 min after dehorning, a significant (P<0.01) decrease in PGE2 concentration was observed in flunixin-treated animals compared with control calves. Moreover, circulating log PGE2 concentrations were inversely proportional to log flunixin concentrations (R(2)=0.75; P<0.0001). None of the other drugs significantly affected blood PGE2 levels. Further assessment of oral meloxicam and gabapentin in established pain models is required to formulate science based analgesic recommendations to enhance animal well-being after dehorning.

Effects of housing and short-term transportation on hormone and lymphocyte receptor concentrations in beef cattle

The experiment was designed to evaluate the effects of housing system and short-term transportation on the pituitary and adrenal response and on blood progesterone concentrations of beef cattle. Since the use of steroid hormones in farm animals has been banned in the EU (Council Directive 96/22/EC), it seems important to study the possible modifications in serum progesterone concentrations induced by stress in cattle. Thirty-two, 6 months old male Piedmontese beef cattle (16 reared in a littered loose house, Group A, and 16 housed in a littered tying stall barn, Group B) were blood sampled at T1 (6 months old), T2 (12 months old), T3 (18 months old, before transportation to the slaughterhouse) and T4 (after transportation to the slaughterhouse) in order to measure hormonal concentrations and lymphocyte glucocorticoid (GR) and β-adrenergic (β-AR) receptor concentrations. Circulating hormone concentrations were measured using commercial radioimmunoassay kits, whereas lymphocyte receptor density was determined through binding assays. In beef cattle housed in tie stall barn a significant increase in serum cortisol concentration was observed at T3, whereas there was no effect of the housing system on blood progesterone concentrations. Short-term transportation caused a significant increase in blood cortisol and catecholamine concentrations in both groups, whereas lymphocyte GR and β-AR significantly decreased in Group A. Our data confirm the activation of the hypothalamic-pituitary-adrenal axis and the catecholaminergic system in short-term transportation and suggest that the stress-induced increase in circulating progesterone concentrations does not exceed the limit established by pending legislation.

Identification of Functional α-Adrenoceptor Subtypes in the Bovine Female Genital Tract During Different Phases of the Oestrous Cycle

The concentration and functionality of the α-adrenoceptor (α-AR) subtypes in the genital tract of cyclic heifers were investigated. In each tissue sample, a single class of α1-ARs was observed, whereas two distinct classes of α2-ARs were discriminated: low-affinity (LA) and high-affinity (HA) α2-ARs. Statistical analysis showed the presence of significantly (p < 0.05) higher concentrations of all α-AR subtypes in the follicle than in the corpus luteum. No significant differences were found in the ovary or myometrium between the luteal and follicular phases. In the ovary, the density of α1-ARs was significantly (p < 0.05) higher than that of α2-ARs. By contrast, there were significantly (p < 0.05) more α2-ARs than α1-ARs in the myometrium. As far as α2-ARs are concerned, LA α2-ARs were significantly (p < 0.05) higher than HA α2-ARs in all tested tissues. Competition studies suggested that the rank order of potency of antagonists for α1-ARs was prazosin > phentolamine > yohimbine, whereas for α2-ARs the order of potency was yohimbine ≥ phentolamine > prazosin. Functional assays performed on myometrium showed that noradrenaline, phenylephrine and clonidine elicited concentration-dependent contractions only in dioestrus and pro-oestrus preparations and that clonidine was more effective than phenylephrine as a contractile agent. It appeared that there were no significant modifications in α-AR affinity or concentration during the different stages of bovine oestrous cycle, whereas the uterine spontaneous activity and the responsiveness to α-adrenergic stimulation was strongly influenced by hormonal levels. The modifications of uterine contractility observed during the oestrous cycle may be related to modifications induced in the transductional mechanisms of α-ARs.

Distribution of cytosolic oestrogen and progesterone receptors in the genital tract of the mare

The distribution of oestrogen and progesterone receptors in the equine genital tract was investigated by means of a modified dextran-coated charcoal method on samples collected from the vagina, the cervix and the uterus of 30 healthy adult Polish mares, divided into two groups on the basis of their serum progesterone levels. The concentrations of oestrogen and progesterone receptors were significantly (P < 0.05) lower in the vagina and the cervix than in the uterus, in agreement with data from human beings, cattle and pigs, which showed that the highest concentrations of oestrogen and progesterone receptors were localised respectively in the body and in the horns of the uterus.

Effects of a β2-agonist (clenbuterol) on cultured human (CG-5) breast cancer cells

Abstract In order to gain further knowledge about the possible oestrogen-like activities of clenbuterol (a gb2-adrenergic drug illegally used as partitioning agent in food producing animals), we treated a hormone dependent human breast cancer cell line (CG-5) with different concentrations of the drug (10 −3 M to 10 −8 M). The effects of clenbuterol and oestradiol on cell proliferation were compared. Both oestradiol and clenbuterol, at low concentrations (10 −7 M and 10 −8 M) stimulated cell proliferation, but the effects of clenbuterol were less marked and significant. Probably clenbuterol elicited cell proliferation through a different mechanism, since it did not affect the cellular oestrogen receptor concentration. Clenbuterol failed in binding to the high affinity oestrogen receptors present in the CG-5 cells. As the β-adrenergic receptors and the susceptibility to their stimulation have been recently demonstrated in vivo and in vitro in many tumour and normal cells, it is reasonable to suppose that clenbuterol may induce cell proliferation through β-adrenergic stimulation.

In vitro and ex vivo pharmacodynamics of selected non-steroidal anti-inflammatory drugs in equine whole blood.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenases (COX), and the inhibition of COX-2 rather than COX-1 can limit the onset of NSAID-related adverse effects. The pharmacodynamic properties of eltenac, naproxen, tepoxalin, SC-560 and NS 398 in healthy horses were investigated using an in vitro whole blood assay. To predict COX selectivity in clinical use, eltenac and naproxen were also studied ex vivo after intravenous administration. SC-560 acted as a selective COX-1 inhibitor, tepoxalin as a dual inhibitor with potent activity against COX-1, and NS 398 as a preferential COX-2 inhibitor. Eltenac was a preferential COX-2 inhibitor in vitro but un-selective in the ex vivo study. Naproxen maintained its non-selectivity both in vitro and ex vivo. These findings have demonstrated that in vitro studies may not accurately predict in vivo NSAID selectivity for COX and should be confirmed using an ex vivo whole blood assay.

Effects induced by exercise on lymphocyte β-adrenergic receptors and plasma catecholamine levels in performance horses.

The effect of dynamic exercise on complete blood cell count, lymphocyte β-adrenergic receptor and plasma catecholamine (adrenaline and noradrenaline) levels in horses performing different disciplines were investigated during rest and after exercise. Blood samples were collected from jumping horses (n=6), Arabian Endurance horses (n=6) and Standardbred trotters (n=6) before and immediately after competition. Dynamic exercise caused a significant increase in red blood cell count (Standardbred trotters: P=0.0012), haemoglobin concentration (jumping horses: P=0.001; Standardbred trotters: P=0.01), haematocrit percentage (Standardbred trotters: P=0.005), neutrophil percentage (jumping horses: P=0.0003), lymphocyte percentage (jumping horses: P=0.0003), monocyte percentage (Standardbred trotters: P=0.0008), lymphocyte β-AR numbers (jumping horses: P=0.01; Arabian Endurance horses: P=0.016; Standardbred trotters: P=0.05), plasma adrenaline concentration (Standardbred trotters: P=0.0001) and plasma noradrenaline levels (Standardbred trotters: P=0.003). It is concluded that acute increases in plasma catecholamine concentrations depended on the exercise performed and may induce up-regulation of β-AR in equine lymphocytes. However, the exact mechanism of β-AR up-regulation still remains unclear.

Comparative liver accumulation of dioxin-like compounds in sheep and cattle: Possible role of AhR-mediated xenobiotic metabolizing enzymes.

PCDDs, PCDFs, and PCBs are persistent organic pollutants (POPs) that accumulate in animal products and may pose serious health problems. Those able to bind the aryl hydrocarbon receptor (AhR), eliciting a plethora of toxic responses, are defined dioxin-like (DL) compounds, while the remainders are called non-DL (NDL). An EFSA opinion has highlighted the tendency of ovine liver to specifically accumulate DL-compounds to a greater extent than any other farmed ruminant species. To examine the possible role in such an accumulation of xenobiotic metabolizing enzymes (XME) involved in DL-compound biotransformation, liver samples were collected from ewes and cows reared in an area known for low dioxin contamination. A related paper reported that sheep livers had about 5-fold higher DL-compound concentrations than cattle livers, while the content of the six marker NDL-PCBs did not differ between species. Specimens from the same animals were subjected to gene expression analysis for AhR, AhR nuclear translocator (ARNT) and AhR-dependent oxidative and conjugative pathways; XME protein expression and activities were also investigated. Both AhR and ARNT mRNA levels were about 2-fold lower in ovine samples and the same occurred for CYP1A1 and CYP1A2, being approximately 3- and 9-fold less expressed in sheep compared to cattle, while CYP1B1 could be detectable in cattle only. The results of the immunoblotting and catalytic activity (most notably EROD) measurements of the CYP1A family enzymes were in line with the gene expression data. By contrast, phase II enzyme expression and activities in sheep were higher (UGT1A) or similar (GSTA1, NQO1) to those recorded in cattle. The overall low expression of CYP1 family enzymes in the sheep is in line with the observed liver accumulation of DL-compounds and is expected to affect the kinetics and the dynamics of other POPs such as many polycyclic aromatic hydrocarbons, as well as of toxins (e.g. aflatoxins) or drugs (e.g. benzimidazole anthelmintics) known to be metabolized by those enzymes.

In vitro toxicity and formation of early conjugates in Caco-2 cell line treated with clenbuterol, salbutamol and isoxsuprine.

SummaryCaco-2, a human intestinal cell lines able to differentiate in long-term culture, has been used to assess the cytotoxicity of the β-agonists clenbuterol, salbutamol and isoxsuprine, also used at high doses to obtain lean meat in food producing animals, and to investigate the eventual in vitro formation of early conjugates of these compounds. For this purpose, the cells have been characterized for the activity of UDP-glucuronyltransferase, which is present and increase in the differentiated cells, and for the β-receptors’ binding characteristics, which are those of β1 and β2 subtypes. Isoxsuprine was shown to be the most toxic, followed by clenbuterol and salbutanol. Conjugates have been observed after incubation of the cells both with the lowest isoxsuprine and the highest salbutamol concentrations. No conjugates were detected in the case of clenbuterol.