Precision-cut liver slices as a model for assess hepatic cellular response of chitosan-glutathione nanoparticles on cultures treated with zilpaterol and clenbuterol.

Zilpaterol and clenbuterol are two ß-adrenergic agonist drugs used in animal production. Both drugs have anabolic effects with advantages on carcass yield. Meanwhile, zilpaterol is approved for animal feed in authorized countries. Clenbuterol is a banned substance due to the risk of toxicity; however, it is still being used in unknown dose levels in many farm species. Therefore, the use and abuse of these substances should be closely monitored, considering the clenbuterol ability and the not proved yet of zilpaterol to produce reactive oxygen and nitrogen species. Regarding glutathione which is the main intracellular antioxidant plays detoxification functions on liver metabolism; in this work, it is our interest to know the capacity of chitosan-glutathione nanoparticles (CS/GSH-NP) as a complementary source of exogenous GSH to modify the oxide-reduction status on bovine precision-cut liver slice cultures (PCLS) exposed to clenbuterol and zilpaterol. A single drug assay was performed in first instance by adding clenbuterol, zilpaterol, chitosan nanoparticles (CS-NP), and CS/GSH-NP. Then combinate drug assay was carried out by testing clenbuterol and zilpaterol combined with CS-NP or CS/GSH-NP. The results showed that both ß-adrenergic agonists modify in a dose-dependent manner in oxide-reduction response through ROS generation. The activity or content of glutathione peroxidase activity, intracellular GSH, gamma glutamyl-transpeptidase, aspartate aminotrasnferase and alanine aminotrasnferase were modified. The exogenous GSH delivered by nanoparticles could be used to modulate these markers.

Mutagenicity and DNA-damaging potential of clenbuterol and its metabolite 4-amino-3,5-dichlorobenzoic acid in vitro.

The aim of this study was to evaluate in vitro toxicity of clenbuterol and its metabolite 4-amino-3,5-dichlorobenzoic acid. Cytotoxicity and pro-oxidative effect of both compounds were studied on human colon adenocarcinoma cell line SW 480. No significant cytotoxic effect of either compound was observed. Results of an Ames test on Salmonella typhimurium did not indicate mutagenic activity of clenbuterol on TA 98 and TA 100 strains, regardless of metabolic activation. Potential mutagenic effects of the highest clenbuterol concentration (2500 ng/ml) were observed on the TA 1535 strain. The obtained results of alkaline comet assay on isolated human lymphocytes suggested that both compounds induced an increase of primary DNA damage in a concentration-dependent manner. 4-ADBA was a slightly more potent inducer of primary DNA damage as compared to clenbuterol. Chromosomal aberration analysis showed that clenbuterol caused a statistically significant increase in the total number of aberrant cells only at the highest concentration tested (3% vs. 0.7% in the negative control). The results of this study might represent a solid frame for designing and planning future studies with both compounds, which should further clarify their mechanisms of action and genotoxic/cytogenetic effects relevant for human risk assessment.

Time course in calpain activity and autolysis in slow and fast skeletal muscle during clenbuterol treatment.

Calpains are Ca2+ cysteine proteases that have been proposed to be involved in the cytoskeletal remodeling and wasting of skeletal muscle. Cumulative evidence also suggests that ß2-agonists can lead to skeletal muscle hypertrophy through a mechanism probably related to calcium-dependent proteolytic enzyme. The aim of our study was to monitor calpain activity as a function of clenbuterol treatment in both slow and fast phenotype rat muscles. For this purpose, for 21 days we followed the time course of the calpain activity and of the ubiquitous calpain 1 and 2 autolysis, as well as muscle remodeling in the extensor digitorum longus (EDL) and soleus muscles of male Wistar rats treated daily with clenbuterol (4 mg·kg-1). A slow to fast fiber shift was observed in both the EDL and soleus muscles after 9 days of treatment, while hypertrophy was observed only in EDL after 9 days of treatment. Soleus muscle but not EDL muscle underwent an early apoptonecrosis phase characterized by hematoxylin and eosin staining. Total calpain activity was increased in both the EDL and soleus muscles of rats treated with clenbuterol. Moreover, calpain 1 autolysis increased significantly after 14 days in the EDL, but not in the soleus. Calpain 2 autolysis increased significantly in both muscles 6 hours after the first clenbuterol injection, indicating that clenbuterol-induced calpain 2 autolysis occurred earlier than calpain 1 autolysis. Together, these data suggest a preferential involvement of calpain 2 autolysis compared with calpain 1 autolysis in the mechanisms underlying the clenbuterol-induced skeletal muscle remodeling.

Clenbuterol induces cardiac myocyte hypertrophy via paracrine signalling and fibroblast-derived IGF-1.

The ß(2)-selective adrenoreceptor agonist clenbuterol promotes both skeletal and cardiac muscle hypertrophy and is undergoing clinical trials in the treatment of muscle wasting and heart failure. We have previously demonstrated that clenbuterol induces a mild physiological ventricular hypertrophy in vivo with normal contractile function and without induction of α-skeletal muscle actin (αSkA), a marker of pathological hypertrophy. The mechanisms of this response remain poorly defined. In this study, we examine the direct action of clenbuterol on cardiocyte cultures in vitro. Clenbuterol treatment resulted in increased cell size of cardiac myocytes with increased protein accumulation and myofibrillar organisation characteristic of hypertrophic growth. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed elevated mRNA expression of ANP and brain natriuretic peptide (BNP) but without change in αSkA, consistent with physiological hypertrophic growth. Clenbuterol-treated cultures also showed elevated insulin-like growth factor I (IGF-1) mRNA and activation of the protein kinase Akt. Addition of either IGF-1 receptor-blocking antibodies or LY294002 in order to inhibit phosphatidylinositol 3-kinase, a downstream effector of the IGF-1 receptor, inhibited the hypertrophic response indicating that IGF-1 signalling is required. IGF-1 expression localised primarily to the minor population of cardiac fibroblasts present in the cardiocyte cultures. Together these data show that clenbuterol acts to induce mild cardiac hypertrophy in cardiac myocytes via paracrine signalling involving fibroblast-derived IGF-1.

Effect of anabolics on bovine granulosa-luteal cell primary cultures.

Granulosa cell tumours are observed with increased frequency among calves slaughtered in Northern Italy. The use of illegal anabolics in breeding was taken into account as a cause of this pathology. An in vitro approach was used to detect the possible alterations of cell proliferation induced by anabolics on primary cultures of bovine granulosa-luteal cells. Cultures were treated with different concentrations of substances illegally used in cattle (17beta-estradiol, clenbuterol and boldione). Cytotoxicity was determined by means of MTT test, to exclude toxic effects induced by anabolics and to determine the highest concentration to be tested. Morphological changes were evaluated by means of routine cytology, while PCNA expression was quantified in order to estimate cell proliferation. Cytotoxic effects were revealed at the highest concentrations. The only stimulating effect on cell proliferation was detected in boldione treated cultures: after 48 h treated cells, compared to controls, showed a doubled expression of PCNA. In clenbuterol and 17beta-estradiol treated cells PCNA expression was similar to controls or even decreased. As the data suggest an alteration in cell proliferation, boldione could have a role in the early stage of pathogenesis of granulosa cell tumour in cattle.

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

Caco-2, human intestinal cell line able to differentiate in long-term culture, has been used to assess the cytotoxicity of the beta-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 increases in the differentiated cells, and for the beta-receptors' binding characteristics, which are those of beta 1 and beta 2 subtypes. Isoxsuprine was shown to be the most toxic, followed by clenbuterol and salbutamol. 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.

[Clenbuterol and fenoterol--animal experiment comparison of 2 tocolytic agents (with special reference to cardiovascular side effects)]. / Clenbuterol und Fenoterol--Tierexperimenteller Vergleich zweier Tokolytika (mit besonderer Berücksichtigung der kardiovaskulären Nebenwirkungen).

During animal experiments two substances, which are used for tocolysis (Fenoterol and Clenbuterol) have been compared regarding their tocolytic efficiency (determinations of myometrial cyclic AMP after long term treatment of the pregnant rat), their effects upon myocardial high energy phosphates (determinations of maternal myocardial high energy phosphates as well as of maternal and fetal myocardial cyclic AMP after long term treatment of the pregnant rat) and upon the hemodynamic situation (acute experiments with thoracotomized dogs). While significant hemodynamic derangements could be stated when using Fenoterol, no significant evidence for such alterations could be found during Clenbuterol administration during acute experiments. Determinations of myocardial high energy phosphates however reflected an augmented myocardial workload, both after Fenoterol and Clenbuterol administration. As by means of myometrial cyclic AMP determinations Clenbuterol proved to be at least as efficient as Fenoterol, concerning the tocolytic effect, Clenbuterol can be recommended as an oral tocolytic because of its pharmaco-cinetic advantages and the encouraging results from our hemodynamic investigations. According to results from chronical experiments an additional cardioprotection by means of magnesium substitution and eventually beta 1-blockade is still recommended.

Toxicology of mabuterol.

The acute, subacute and chronic toxicity of dl-1- (4-amino-3-chloro-5-trifluoromethyl-phenyl)-2-tert.-butyl-amino-ethanol hydrochloride (mabuterol), a new beta 2-selective adrenoceptor stimulant, was studied in mouse, rat, rabbit and dog. The acute oral LD50 was between 199.9 and 319.3 mg/kg in all four species used and did not differ significantly between the sexes. Intravenous LD50 was between 18.3 and 51.1 mg/kg (four species), i.p. between 60 and 78.3 mg/kg and s.c. between 113 and 125.7 mg/kg (mouse and rat only). In subacute and chronic studies in Wistar rats, mabuterol was dosed by gavage at 0, 10, 20, 40, 80 and 120 mg/kg for 1 month, and 0, 1, 2.5, 10 and 40 mg/kg for 3 (interim) and 6 months or in food at 0, 2.5, 10 and 40 mg/kg for 6 (interim) and 12 months. Beagle dogs were dosed by gelatine capsule with 0, 0.05, 0.5, 5 and 50 (25) mg/kg for 6 (interim) and 24 months. In rats, 1 and 2.5 mg/kg were well tolerated. At 10 mg/kg and above, irritability and hypersalivation were seen dependent on dose. Two males and one female given 120 mg/kg died from the test substance. In dogs, 0.05, 0.5 and 5 mg/kg were well tolerated. 50 mg/kg caused vomiting, diarrhoea, tonic-clonic convulsions and increased salivation. One male had to be killed because of severe convulsions. At term, 3 of 56 female rats of the one-year study had benign mesovarian leiomyomas. No other substance-related changes were discovered but all findings belonged to the spontaneous pathology of rat and dog, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)