ࡱ> ac`5@#bjbj22.<XXzzzzzzzDFh~~~~~~~RFTFTFTFTFTFTF$GRIpxFz~~~~~xFzz~~FEEE~$z~z~RFE~RFE,EEzzE~\ 5 r`AEFLF0FEKJ*EjKJET6zzzzKJzE<~~E~~~~~xFxFE  EMBED Word.Document.8 \s  INTRODUCTION Clenbuterol ((4-Amino-3,5-dichlorophenyl)-2-tert-butyl-aminoethanol hydrochloride], a (2-adrenergic agonist (BAA), is used in the treatment of pulmonary diseases in human and veterinary medicine. In recent years, this pharmacologically active substance has been illegally used at 5- to 10-fold therapeutic doses as a repartitioning agent to improve the performance of food-producing animals (1). Many countries have imposed total ban on the use of clenbuterol in food-producing animals, since its abuse may have adverse effects on animal welfare and induce pharmacotoxicologic risks for consumer health (2).Previously published results have shown that persistance of clenbuterol in the liver is much higher that in other edible tissues and detectable for up to 2 weeks after withdrawal of the drug from animal's diet (3). Because of this, and the fact that liver with its metabolic, detoxicating, secretory and excretory function is extremely sensible to toxic substances, the aim of our study was to investigate, using biochemical and histopathologic analysis, whether clenbuterol residues in the liver after repeated administration at a growth-promoting dose caused an alteration in the pig hepatic function. MATERIAL AND METHODS Chemicals Clenbuterol hydrochloride standard was obtained from Sigma-Aldrich-Chemie, Steinheim, Germany. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were purchased from ThermoTrace, Melbourne, Australia. (-Glutamyltransferase (GGT) was purchased from Trace, Melbourne, Australia. All other chemicals were analytical-grade commercial specimens. Animals and sampling procedure The experiments were carried out in 12 female pigs (6 control and 6 treated) of a known breed, aged 6- 7 months, body mass 80-100 kg. The pigs were given 10 (g clenbuterol/kg body mass, intravenously, twice daily from day 1 (at the onset of estrus) to day 25. On day 25, blood samples were collected , kept at room temperature for 90 min to allow clot retraction before centrifugation at 2500 x g for 15 min and were stored in glass vials at -150C before analysis. The pigs were sacrificied according to standard slaughter-house procedures. The livers were collected for residual clenbuterol determination and for macroscopy and histopathology.The experimental protocol was designed according to the Act on Animal Welfare, as stated in the Official Gazette of the Republic of Croatia No.19/1999. Assays For residual clenbuterol determination, liver samples were homogenized, extracted with tertiary butylmethyleather, cleaned up on RP-18 cartridges (Supelco, Bellefonte, USA), eluated with methanol and chromatographed by high-performance liquid chromatography (HPLC)(Perkin Elmer).The chromatographic condition for HPLC were: binary LC pump 250, LC-95 UV/VIS Perkin Elmer detector and PE Nelson spectromonitor model 1020; column-LiChrospher 60-RP Select B 200 x 4 mm, 5 (m; mobile phase : methanol-buffer (20 mM KH2PO4, 30 (M EDTA, pH 3.9)- (25:75 v/v) at flow rate of 1 ml/min at 250 C. UV detection was at 244 nm. Serum activities of ALP, ALT and AST were determined using TermoTrace reagent kits and methods. The methods are based on the recommendation of the International Federation of Clinical Chemistry. Serum GGT activity was measuring by the method of Szasz (4). Preparation of liver for histologic studies The livers were fixed in 10 % phosphate-buffered formalin and paraffin-embedded 5 (m thick sections were stained with hematoxylin and eosin. Data analysis Results are presented as mean ( SE. Student's t-test was used to evaluate the significance of difference between the means, with the statistical limit set at < 0.05. RESULTS Determination of residual clenbuterol in pig livers after cessation of administration (day 25) by HPLC The intra-assay variation of the method is shown in Table 1. Good recoveries (> 80%) were obtained for both levels of fortification. The intra-assay results show acceptable variation within a single assay samples fortified at 30 and 50 ng/g (RSD<10 %). The concentration of clenbuterol detected (day 25) varied from 32.42 to 58.30 ng/g in liver samples of six treated pigs (Table 2.). Macroscopic and histopathologic findings There were no significant macroscopic differences in liver between the experimental and control pigs. On day 25 after cessation of clenbuterol administration, histopathologic alterations in all livers from experimental group of animals manifested as hepatocyte degeneration and vacuolization, and hyperplastic cholangitis (Figure 1.). Effects of clenbuterol administration on serum enzyme activities Effects of the repeated intravenous administration of clenbuterol in a growth-promoting dose on pig serum GGT,ALT, AST, and ALP activities determined after the last dose (day 25) are shown in Figure 2. The activities of these enzymes were compared with the reference ranges adopted from Boyd (5). The results howed significantly increased serum activities of ALP (p<0.01) and ALT (p<0.01), and slightly decreased serum AST although still within the normal values. Serum activity of GGT was increased but the difference was not statistically significant. CONCLUSIONS In our study, the increased activity of ALT as a cytosolic enzyme in correlation with the histopathologic findings pointing to hepatocyte degeneration suggested the adverse effect of clenbuterol to occur at the cellular level. On the other hand, the increased serum activity of ALP a cholestatyc enzyme associated with cholestatic lesions suggested clenbuterol interference with bile secretion. In conclusion, repeated administration of a growth-promoting dose of clenbuterol to female pigs led to the accumulation of clenbuterol in the liver as a edible tissue. Biochemical and histopathologic analysis used in the evaluation of the effect of clenbuterol on hepatic function suggested that clenbuterol residues in the liver could adversely affect the functional activities of this parenchymatous organ, thus also the animal and indirectly human health. REFERENCES 1. Mersmann HJ: Potential mechanisms for repartitioning of growth by -adrenergic agonists. In: Animal growth regulation Ed.Campion DR, Hausman GJ & Martin RJ, Plenum Press, New York, pp. 337-357, 1989. 2. Martinez-Navarro JF: Food poisoning related to consumption of illicit (-agonists in liver. Lancet 336: 1311, 1990. 3. Elliott CT, McEvoy JDG, McCaughey WJ et al: Improved detection of the (-agonist clenbuterol by analysis of retina extracts. Vet Rec 132: 301-302, 1993. 4. Szasz G: A kinetic photometric method for serum (-glutamyltranspeptidase. Clin Chem.15: 124-136, 1969. 5. Boyd JW: The interpretation of serum biochemistry test results in domestic animals. 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