196,182 research outputs found
Effects of illicit drug treatments upon the cattle liver cytochrome P4503A (CYP3A) dependent hydroxylation of testosterone and protein expression.
No full textDespite the ban at the European Union level (Dir. 88/146/EEC), growth promoters (GPs) are still illegally used in cattle, alone or in combination, to improve carcass quality and meat performances (Courtheyn, 2002). The cytochrome P4503A (CYP3A) is the P450 isoform responsible for the oxidative metabolism of most drugs clinically used as well as of endogenous steroid hormones (Dacasto, 2005). In the present study the effects of several GPs administered alone or in combination on the cattle liver CYP3A, in terms of catalytical activity and protein expression levels, were investigated. Liver microsomes, obtained from the caudate lobe of control or GPs-treated cattle by differential centrifugation (Nebbia, 2003) were incubated, at 37°C for 10 min, with 250 μM testosterone (TST) and its 6β- and 2β-hydroxy-metabolites were separated by HPLC (Purdon, 1997). The CYP3A protein levels were measured by immunoblotting, by using mono- or polyclonal antibodies directed toward the respective rat and/or human isoform (Dacasto et al., 2005). In veal calves, a significant reduction in CYP3A-dependent TST 6β- and 2β-hydroxylation was observed in animals treated with a cocktail of 17β-oestradiol (17βOE), clenbuterol and dexamethasone (DEX); quite surprisingly, the same result was obtained with DEX given alone. These results were confirmed also at the protein level. On the contrary, mixtures of 17βOE-boldenone (BOL), 17βOE-TST or BOL-boldione (ADD) never significantly altered the liver CYP3A expression of treated animals. In beef cattle, the illicit DEX administration induced CYP3A, whereas animals administered with endogenous steroids or BOL precursors (dehydroepiandrosterone and ADD, respectively), given alone or in association, never shown differences in the liver CYP3A expression. Present results demonstrate that GPs are likely to differentially modulate CYP3A expression in terms of catalytic activity or protein expression, corroborating data obtained with other P450 isoforms (i.e. CYP1A and CYP2C: Dacasto, in press). Therefore, these ones cannot be used as a screening test for illicit drug treatments in cattle. Ongoing studies are currently envisaged in our lab to investigate the effects of GPs at the gene expression level, by using quantitative real-time PCR and/or drug metabolism microarray.
The financial support of Regione Piemonte, Regione del Veneto and Istituto Zooprofilattico Sperimentale delle Tre Venezie is gratefully acknowledged
IlLEGAL USE OF GROWTII PROMOTING AGENTS AND ALTERATIONS IN BOVINE FEMALE REPRODUCTIVE SYSTEM
No full textIntroduction·
The P1-agonist clenbuterol is a drug that finds wide application in. veterinary medicine as
. bronchodilator and tocolytic agent. In recent years, ìt has been more and more utiIized as repartitioning agent to improve the performance of meat animals. In faci. clenbuterol long-term administration improves growth rate, reduce fat deposition and increase protein accretion in cattle, pigs and poultry. From this point of view, clenbuterol treatment should fulfil producers and consumers demands (1). Thus, in Italy, veal calves are reasonably suspected to be systematically treated with P-agonists in order to stimulate growth. Nevertheless, a field study carried out on
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regularly slaughtered veal calves. revealed a ratber constant involvement of the female genital tract consisting in anatomical and histopathological changes associated with biochemicallesions (23). The alterations present in the female reproductive system of veal calve s, sequestrated by the Judicial Authority for illegal treatment with clenbuterol, were investigated. In the urine of six male belonging to the same herd, clenbuterol were found in not allowed amounts.
Materials and methods The histological investigations were performed on the uteri, ovaries and major vestibolar glands of 15 crossbred veal calves, 2-4 months old. Tissue samples were fIxed in buffered formalin (10%) and tben paraffIn-wax embedded sections were stained by routine methods (Haematoxilin-eosin, Van Gieson, Azan, Pas-reaction). Major vestibolar glands were processed using more specifIc techniques (Alcian-Pas pH 2.6 and Alcian pH 1). Just after slaughtering, tissue samples (about 1 g) were dissected from the vagina (A), the body (B), and the uterine horns (C) and then frozen at -80"C in order to assay cytosolic estrogen and progesterone receptor concentrations using a modified dextrancoated charcoal method (3). The results obtained by the Scatchard analysis, are expressed as femtomoles of specifIcally bound hormone per mg of protein (mean values±S.E.M.). The hormonal status, was evaluated on serom samples obtained from clotted blood drawn from the jugular vein of each animaI. Estradiol and progesterone serom level were measured using RIA techniques (Estradiol Double Antibodl and Progesterone Coat-A-CountO DPC Los Angeles, USA).
----,1 Results The lumen of the vagina and of the uteros was constantly filled with an abnormal collection of transparent mucus, and in all examined animals
r----· l
microcystic ovaries were presento From the histological point of view, the cervical part of the uteros showed pluristratification of the endometrial epithelium, frequently associated with squamous metaplasia of the inner layer. In the oldest calves, glandular hyperplasia and hypersecretion were detected. Endometrial cysts were also presento The major vestibular glands showed hyperplasia of ductal and glandular epithelium with ectasic lumina filled with
·0 I I J~~P~w_...I._~~~-L~~~' t
Pas positive or Alcian-Pas positive material;
r scattered foci of mononuclear cells and metaplastic transformation were occasionally found. In the ovaries, atretic tertiary folli cles associated witb normal ones were seen. The number of uterine estrogen and progesterone receptors reached high concentrations mainly in tbe
Figure 1. Uterine estrogen and progesterone uterine body (B) and horns (C) (estrogenreceptor concentrations (mean values±S.E.M.)
receptors, B: 480± 98, C: 487 ± 130; progesterin veal calves. A=vagina, B=body, C=horns one receptors, B: 632 ± 118, C: 1137 ± 292). In (n= 15) the vagina (A) only the estrogen receptors are
particularly elevated (A: 123+22), whereas progesterone receptor levels are normal (Fig. 1). RIA investigations revealed that neitber traces of estradiol nor progesterone were present in the examined sera.
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Diseussion The histological pictures, reveal a morphological and physiological status incompatible with the immature age of the animals. On the other hand, these findings suggest a condition of exogenous stimulation by growth-promoting or anabolic agents. Moreover, the high concentrations of estrogen and progesterone receptors at uterine level, when compared with normal values (4), also indicate an abnormal disposition of the receptorial status. It is well established that estrogenic treatments induce steroid receptors syntesis, but recently, clenbuterol treatment has been related with uterine estrogen and progesterone receptors increase in rat and pigs (5), without affecting or at least inhibiting hormonal serum levels.
Reterences
1.
VanbeHe M., 1990, New technology governing nutrient partÌtioning in meat animals. Atti Conf. Int. Sanita Prod. Bovina Mediterraneo, 1, 459.
2.
Girardi C., Badino P., Re G., Dacasto M., Biolatti 8., Brusa F., Di Carlo F., 1990, Stato recettoriale e reperti anatomo ed istopatologici del tratto genitale femminile in vitelli a carne bianca. Atti Soc. Il. Buiatria, 22, 375.
3.
Di Carlo F., Racca S., Conti G., Gallo E., Muccioli G., Sapino A., Bussolati G. 1984, Effeets of long-term administration of high doses of medroxyprogesterone aeetate on hormone receptors and target organs in the female rat. J. Endocr. 103, 'll37.
4.
Re G., Badino P., Conti G., Dacasto M., Di Carlo F., Girardi c., 1989, Distribution of eytoplasmic estrogen (ER) and progesterone (PgR) receptors in bovine genital traet. Pharmaool. Res., 21, suppl.1, 79.
5 Re G., Badino P., Tartari E., Biolatti B., Di Carlo F., Girardi C., 1990, Clenbuterol long-term administration in fmishing female pigs. Note 1: effeets on oestrogen and progesterone receptor distribution and ooncentration in genital tract. Schweiz. Arch. Tierheilk., 132, 455
Expression of liver cytochrome P450 drug metabolising enzymes in different meat cattle breeds
No full textIntroduction. The cytochrome P450 (P450) superfamily constitutes a multigene membrane-bound enzyme system which catalyses oxidations of both xenobiotics and relevant endogenous compounds (1). Several constitutional factors (gender, age, species, strain, pathological and physiological conditions) might contribute, together with induction and inhibition phenomena (which are classified as exogenous or xenobiotic-dependent factors) to the modulation of the overall biotransformation capacity (2). In a previous study, differences in the expression of the liver cytochrome P4503A subfamily have been reported in Limousine and Piedmontese cattle breeds (3). As an in-depth study, the expression of main P450-dependent drug metabolising enzyme (DME) activities (both at the catalytic and protein expression levels) were measured in the liver of three different meat cattle breed.
Materials and Methods. Microsomal subcellular fractions were prepared, from the liver of male Charollais (C: n=10), Blonde d’Aquitaine (B: n=7) and Piedmontese (P: n=8) beef cattle, about 13-18 months old. Total P450 content and main P450-dependent DME activities were measured, by using known model substrates, according to previously published protocols (4, 5). Furthermore, the cytochrome P4501A (CYP1A), 2B (CYP2B), 2C (CYP2C) and 3A (CYP3A) apoprotein levels were measured, in pooled microsomes, by immunoblotting (using polyclonal antibodies directed toward the respective rat and/or human P450 isoforms) as reported elsewhere (3). The densitometric analysis of visualised bands was performed by ImageJ 1.34s, NIH, Bethesda, MD, USA). Data statistical analyses was performed by using the analysis of variance (ANOVA), followed by a post-test (Tukey-Kramer multiple comparisons test: GraphPad Instat® 3.06 for Windows, GraphPad Software Inc., San Diego, CA, USA).
Results. Significant differences in the total liver P450 content were never found among breeds. Lower CYP1A, CYP2B and CYP2E1 DME activities were noticed in P (P<0.05, P<0.01, P<0.001, depending from the substrate used) vs C or A (P<0.05, P<0.001). At the protein level, lower significant CYP2B amounts were found in P and C vs B (P<0.01). No statistically significant differences were ever found in CYP1A, 2C and 3A contents.
Discussion and Conclusion. It is known that several non pharmacogenetic factors such as age, gender, species, disease factors or exposure to environmental pollutants might contribute to the expression and regulation of hepatic P450 in domestic animals (2). It has been otherwise underlined how studies related to the expression of DME in farm animals might be very helpful in food safety assessment and drug registration requirements as well (4, 6). This of particular importance in cattle, which represent an important source of animal-derived food products. In this respect, studies on DME expression in Ruminants have already been published (6-10). Data obtained in the present study demonstrate that, mostly at the catalytical activity level, some differences exist between different cattle breeds, confirming what previously observed in liver CYP3A expression in P vs Limousine cattle (3). Actually, further immunological investigations to complete data for each P450 protein levels have been undertaken in our laboratory; this would permit the evaluation of linear correlation analysis between the individual CYP1A, 2B, 2C and 3A protein amounts and the respective model substrate catalytic activity, within and between the three different breeds. Furthermore, oncoming studies, whose major goal might be the confirmation of these preliminary results also at the gene expression levels, are foreseen.
Reference. Honkakoski P. and Negishi M. Biochem. J. 2000; 347: 321-337. 2. Nebbia C. Vet. J. 2001; 161: 238-252. 3. Dacasto et al. Vet. Res. 2005; 36: 179-190. 4. Nebbia et al. Vet. J. 2003; 165: 53-64. 5. Purdon & Lehmann-McKeman J. Pharmacol. Toxicol. Methods 1997; 37: 67-73. 6. Sivapathasundaram S. et al. Biochem. Pharmacol. 2001; 62: 635-645. 7. Machala M. et al. Arch. Toxicol. 2003; 77: 555-560. 8. Sivapathasundaram S. et al. Toxicology 2003; 187: 49-65. 9. Sivapathasundaram S. et al. Comp. Biochem. Physiol. 2003; 134C: 169-173. 10. Szotáková B. et al. Res. Vet. Sci. 2004; 76: 43-51.
Acknowledgements. This study was supported by a grant from Università degli Studi di Padova (60A08-8213/05: studi sull’espressione degli enzimi farmaco-metabolizzanti nel fegato di bovini appartenenti a razze diverse) to M.D and a two-years post-doctoral fellowship, from Università degli Studi di Torino, to M.C
Use of hepatic and testicular molecular biomarkers to detect growth promoters misuse in cattle: a preliminary application under field conditions
No full textIntroduction. Growth promoters (GPs) are forbidden at the European Community level. Nevertheless, GPs misuse in cattle still represent a major concern. In the past decade an increasing interest toward the set up and validation of molecular biomarkers to be used side by side with official analytical methods has been recorded (Nebbia, 2010). In preceding pilot studies, a number of tissue-specific responsive genes have been identified (Giantin, 2010; Lopparelli, 2011). In this study, these biomarkers were preliminarily tested under field conditions.
Materials and methods. Ninety-five cattle testis and liver aliquots were collected by chance at slaughterhouses, placed in microtubes with RNAlater® and stored at -80°C until use. A robust set of negative controls (44 animals), from earlier pilot studies, were included in the study, too. Total RNA was extracted with TRIzol® Reagent and gene expression profiles measured by using a quantitative Real Time RT-PCR approach (qPCR). Seven and eight target genes were chosen for liver and testis, respectively. Results were elaborated (Hierarchical Clustering, HCL, and Principal Component Analysis, PCA) by using the GenEx software (Berkvist, 2010).
Results. In liver, HCL clustered samples into three main groups, supported by PCA: negative controls and most of random samples were clustered together (“negatives”), while three animals were distinctly grouped in another cluster (“suspects”). Further nine samples, assigned to negatives by GenEx, generated a different cluster; therefore, they were classified as “doubtful”. In testis, three “suspects” and three “doubtful” were identified besides “negatives”. Considering both tissues as a whole, the software identified three “suspects” and two “doubtful”.
Conclusions. This study aimed to test a set of candidate genes and a popular software for qPCR data processing and analysis upon a large number of random samples. The approach allocated samples into three different clusters, representing different expression profiles. Presented data suggest that transcriptome analysis and bioinformatic tools, coupled with a robust database of negative controls, might be helpful for tracking GPs abuse in cattle. Further studies are needed to confirm these promising results.
References. Nebbia C., Urbani A., Carletti M., Gardini G., Balbo A., Bertarelli D., Girolami F., 2010. Novel strategies for tracing the exposure of meat cattle to illegal growth-promoters. The Veterinary Journal, 189: 34-42.
Giantin M., Lopparelli R. M., Zancanella V., Martin P. G., Polizzi A., Gallina G., Gottardo F., Montesissa C., Ravarotto L., Pineau T., Dacasto M., 2010. Effects of illicit dexamethasone upon hepatic drug metabolizing enzymes and related transcription factors mRNAs and their potential use as biomarkers in cattle. Journal of Agricultural and Food Chemistry, 58: 1342-1349.
Lopparelli R. M., Zancanella V., Giantin M., Ravarotto L., Pozza G., Montesissa C., Dacasto M., 2011 Steroidogenic enzyme gene expression profiles in the testis of cattle treated with illicit growth promoters. Steroids, 76: 508-516.
Berkvist A., Rusnakova V., Sindelka R., Garda J. M. A., Sjogreen B., Lindh D., Forootan A., Kubista M., 2010. Gene expression profiling – Clusters of possibilities. Methods, 50: 323-335.
Acknowledgements. Project supported by a grant from Regione del Veneto (Dgr 2888 07/10/2008) to M.D
Efficacy of ivermectin in reducing gastrointestinal nematode fecal egg counts in goats in Burundi.
No full textCross-bred goats in Burundi infested with gastrointestinal nematodes were submitted to fecal investigations and injected subcutaneously with ivermectin. In Experiment 1, goats were treated with 200 mu g kg(-1) bw ivermectin. In Experiment 2, animals were administered twice that dose. In Experiment 3, goats suspected to be resistant to other anthelmintics were treated with 200 mu g kg(-1) bw ivermectin. In Experiment 4, two doses of the same strength were injected with an interval of 7 days. Results demonstrate that 200 mu g kg(-1) bw ivermectin is effective for the control of gastrointestinal nematodes of goats in Burundi; this dosage is also effective against nematodes suspected to be resistant to other anthelmintics. The administration of 400 mu g kg(-1) bw did not induce greater or more prolonged effectiveness percentages. The supposed decrease of ivermectin's residual activity on Day 28 might be avoided by administering two doses with an interval of 7 days. No adverse effects were observed in treated animal
In vitro formation of metabolic-intermediate cytochrome P450 complexes in rabbit liver microsomes by tiamulin and various macrolides
No full textTiamulin and a number of macrolides were evaluated as to their ability in forming metabolic-intermediate (MI) complexes with cytochrome P450 in liver microsomes from rabbits bred for meat production. Complex formation, which occurred only in preparations where the expression of P450 3A was increased as the result of rifampicin pre-treatment and with different kinetics, was in the order tiamulin > erythromycin > TAO approximate to roxithromycin approximate to tylosin and did not take place with tilmicosin and spiramycin. Most of the tested compounds underwent an oxidative N-dealkylation and a good relationship could be found between the rate of N-dealkylase activity in induced preparations and the aptitude in generating MI complexes. Although the results from in vitro studies should be interpreted with caution, it is suggested that the potential for in vivo drug interactions also exists in the rabbit for tiamulin and for four out of the six tested macrolide
Postnatal development of hepatic, hydrolytic and conjugative drug-metabolizing enzymes in female horses
No full textLittle is known about the effects of aging on the hepatic drug metabolizing capacity of horses despite the relatively long lifespan characterizing this species. A wide array of cytochrome P450 (CYP)-dependent monooxygenases, carboxylesterases and transferases were assayed in liver microsomes from 50 female horses in an age range between less than 1 year to over 12 years. Rather unexpectedly, both the CYP content and the activity of NADPH cytochrome c reductase rose as a function of age. Accordingly, a general increasing trend was recorded in the rate of the in vitro metabolism of the substrates reported to be related to CYP2B-, CYP2E- or CYP3A, although, as detected by Western immunoblotting, only the levels of proteins recognized by anti-rat CYP3A- and CYP2B antibodies appeared to increase consistently. Also the carboxylesterases and uridindiphosphoglucuronyl-transferase (UGT) activity toward 1-naphthol displayed a similar trend, glutathione S-transferase accepting 3,4-dichloronitrobenzene as a substrate being the only enzyme activity showing an age-related decline. A positive correlation was also found between liver cadmium content and CYP amount as well as the activities of most monooxygenases (except for those related to CYP1A), carboxylesterases, and UGT. While confirming that a number of enzyme activities are less expressed in foals, our results contradict the general view that the drug metabolizing capacity drops in elder individuals. Although several other factors can influence the kinetics of foreign compounds in aged animals, data from this study may provide insight in understanding possible age-related differences in drug efficacy and the response to toxic substances in horse
Protective effects of quercetin towards aflatoxin B1-induced hepatotoxicity in cattle: a whole transcriptomic in vitro study
No full textIntroduction: Aflatoxin B1 (AFB1) is a natural feed and food contaminant. In the dairy industry, AFB1 and its
derivatives are of concern for the related economic losses and their possible presence in milk and dairy food
products. Oxidative stress is one of the prominent toxic effects of AFB1, thus, dietary supplementation with
natural antioxidants is a valid strategy to mitigate AFB1 toxicity. In this respect, quercetin (QUE) is a promising
bioactive compound. Here we assessed the protective role of QUE in bovine foetal hepatocyte-derived cells
(BFH12) exposed to AFB1, by measuring the QUE impact on AFB1-induced cytotoxicity and related whole-
transcriptional changes.
Methods: to increase cells responsiveness to AFB1, monolayers were pre-treated with 1 nM PCB126 for 24h
and then exposed to 3.6 μM AFB1, alone or in combination with sub-cytotoxic concentrations of QUE (10, 20, 30
μM). Cytotoxicity was then assessed. For the whole-transcriptome study, we exposed cells to 30 μM QUE, 3.6
μM AFB1, or their combination. Cells pre-treated with PCB126 and exposed to DMSO were used as control
(CTRL). Total RNA was isolated, 12 tagged RNA-seq libraries were prepared and sequenced (50 bp single-end).
Reads were quality checked, trimmed and mapped to cow reference genome using validated pipelines. EdgeR
and clusterProfiler were used to identify differentially expressed genes (DEGs) and perform the functional
enrichment analysis, respectively.
Results: 30 μM QUE significantly reduced AFB1-induced cytotoxicity by 13.39%. As to RNA-seq, the differential
expression analysis comparing QUE vs CTRL and QUE+AFB1 vs AFB1 identified 1028 and 1890 DEGs,
respectively. QUE alone affected the expression of genes related to steroids metabolism, inflammation,
immunity, and P450-mediated drug metabolism. When comparing QUE+AFB1 vs AFB1, top upregulated genes
possess antiapoptotic and antioxidant activity (e.g. SEMA5A, TF), while top downregulated genes play a role in
inflammation and cancer progression (e.g. CXCL5, IL6). If comparing these results with the mRNA changes
induced in cells exposed to AFB1 alone, all the top-10 genes upregulated by the cotreatment were inhibited by
AFB1; on the opposite, all the top-10 downregulated genes were induced by AFB1 alone.
Conclusions: we showed the QUE potential to mitigate AFB1-induced toxicity and the underneath molecular
mechanisms. If the beneficial effects will be confirmed in vivo, QUE-supplemented diets might be adopted to
improve health status of cattle exposed to AFB1-contaminated feed
Effects of the repeated oral administration of cimaterol and clenbuterol upon the liver drug metabolizing enzymes activities (DMEs) in the male chick
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