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Giantin, Mery (2008) Effetti di fattori intrinseci ed estrinseci sull'espressione in vivo ed in vitro degli enzimi farmaco-metabolizzanti epatici del bovino. [Tesi di dottorato]

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Abstract (inglese)

Cytochrome P450 superfamily (CYP) comprises an ubiquitous enzyme system, with the highest concentration found in the liver. These enzymes play a crucial role in the metabolism of xenobiotics and endogenous compounds. Their primary role is converting lipophilic compounds to more polar and hydrophilic metabolites by means of oxidative, reductive and hydrolitic reactions. Products of these chemical reactions can then be conjugated with polar endogenous compounds and readily excreted by the organism (Ioannides, 2006). The evaluation of liver biotrasformation pathways in veterinary species, but expecially in cattle, is considered very important, particularly for the presence of potentially harmful residues in foodstuff of animal origin (Sivapathasundaram et al., 2001). Thus, very few informations about bovine metabolism are actually available in literature and they primarily focused on catalytic activities and protein expression data (Sivapathasundaram et al., 2001; Nebbia et al., 2003; Szotáková et al., 2004; Dacasto et al., 2005). Recently, increasing importance has been given to toxicogenomics, the science which studies the correlations between genome structure, activity and toxicological effects of xenobiotics (Aardema and McGregor, 2002). The recent advances in molecular biology have resulted in the possibility to set up innovative, sensitive and specific biomolecular tecniques, that can be successfully applied in Drug Metabolism (DM) studies (Tugwood et al., 2003).
As a consequence, the aim of the present research project consists on the application of some recent biomolecular techniques, as the quantitative Real Time PCR (Q RT-PCR), in order to study the expression and the regulation of some genes involved in bovine DM.
The following methodological approach has been adopted: firstly a bibliographic search to identify genes involved in xenobiotics metabolism and then, collection of the corresponding available mRNA sequences from databases; clonage and sequencing of bovine sequences not yet available; primers design and set up of bovine-specific Q RT-PCR assays; total RNA isolation from cattle liver samples either of control or experimentally treated with illicit growth promoters; finally, the application of Q RT-PCR assays for the relative quantitation of selected transcripts (genes involved in xenobiotics metabolism and/or transcription factors) in: (a) beef cattle belonging to different meat cattle breeds; (b) veal calves and beef cattle illegally treated with dexamethasone (DEX) administered alone per os or intramuscularly, or in association with 17ß-oestradiol (E2: this last treatment has been considered only in beef cattle); (c) in primary cultures of bovine hepatocytes incubated with endogenous/exogenous precursors of steroids and in beef cattle illegally treated with the same molecules, in order to compare in vivo/in vitro effects. Messanger RNA results were successfully compared and/or correlated with catalytic activity data obtained by using marker substrates known as specific for each considered CYP isoform or phase II enzyme, but also with protein expression data (CYP1A, 2B, 2C, 2E and 3A immunoblotting and semiquantitative densitometric analysis).
Partial sequences coding for bovine CYP2B, CYP4A, UGT1A4, RXR?, GR, HNF4? and 17ßHSDII genes (all involved in DM and its regulation) have been cloned, sequenced and submitted to GenBank; furthermore Q RT-PCR assays for a total of 28 transcripts have been designed and set up.
Forementioned assays were applied to evaluate the hepatic biotransformative pattern in 18-20 months old beef cattle belonging to three different meat cattle breeds (Charolais, CH; Piedmontese, PM; Blonde d'Aquitaine, BA). Statistically significant differences (P<0.05 or less) have been noticed in CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2C18, CYP3A4, UGT1A1, UGT1A6, UGT2B17, GSTA1, GSTM1, GSTP1 mRNA expression. In particular, CH presented the lowest mRNA expression for all the forementioned transcripts except for UGT1A6, if compared with the similar expression profile of PM and BA. Results obtained at the pre-transcriptional level were confirmed at the post-translational one by immunoblotting, only in the case of CYP2B and CYP3A: in this respect, protein expression data for these two enzymes demonstrated the same trend among breeds (CH<PM<BA). The in vitro metabolism of specific model substrates for each isoform, instead, did not agree with Q RT-PCR data (except in some instances), but, on the contrary, presented an opposite behaviour: in fact, CH appeared to be the breed gifted of the lower gene expression but of the higher biotransformation capability. This result could be probably ascribed to a more efficient catalytic system or to the presence of polymorphisms. Furthermore, the correlation analysis between single enzyme gene expression and the corresponding catalytic activity has been performed: for some isoforms a good correlation has been evidenced. Present results confirmed the breed as one of the internal factors that might modulate the metabolism in cattle.
The same Q RT-PCR assays have been applied in other two experiences, where 15-18 months old beef cattle have been administered with illicit compounds; the aim of the present approach was to evaluate the effects of these substances on liver metabolism and to identify possible indirect biomarkers of treatment.
In the first experiment, "Marchigiani" beef cattle were treated with DEX per os (TPD group) or intramuscularly (BPD group), whereas in the second study French crossbred cattle were administered DEX alone (Dde group) or in combination with E2 (DEde group). Gene expression data pointed out a significant induction of CYP1A1, CYP1A2 and CYP3A4 mRNA in DEde group, while a consistent inhibition of CYP2B6 e CYP2E1 was noticed in all treated groups. As regards the tested phase II enzymes, GSTA1 was induced in BPD and DEde groups, and SULT1A1 in TPD and DEde ones. Finally, among nuclear receptors, an increase either of CAR and RXR? expression in TPD and DEde groups or ER? (only in DEde group) was noticed. The design and the application of a 28 transcripts PCR-Array of hepatic metabolism in experiments regarding illicit treatments allowed the identification of some potential candidate genes that could be used in the future as indirect biomarkers on field, although only after a validation step.
The same approach (only for some phase I enzymes and corresponding transcription factors) has been used to complete another experiment, in which veal calves were treated only with DEX per os or intramuscularly: the evidenced effects were completely different from other ones collected in beef cattle, probably for physiological differences (age, diet, anatomy and physiology of the gastro-enterical apparatus) that are likely to modulate DEX pharmacokinetics but also for difficulties strictly related with this particular kind of farming (stress conditions and iron-deprived diet). No statistically significant differences were observed in chosen CYPs and nuclear receptors gene expression, except for GR, induced in the group of animals treated intramuscularly with DEX. Furthermore, the CYP3A protein expression was inhibited in both treated groups, confirming the trend observed at both gene expression and catalytic activity levels.
Finally, the effects of some endogenous/exogenous precursors of sexual steroids on some metabolism enzymes and transcription factors gene expression have been evaluated, by comparing one in vitro and one in vivo system. Primary cultures of bovine hepatocytes, incubated for 6 hours with 100 ’µM ADD or DHEA, and 15-18 months old beef cattle ("Valdostana" breed), treated with the same compounds at the dosage of 50 mg/pro capite once a week for 5 weeks, were used. Quantitative Real Time PCR results evidenced that the in vivo treatments with the forementioned molecules did not cause relevant effects (except for PXR and PPAR? inhibition in the group treated with DHEA), confirming both catalytic activities and immunoblotting results. On the contrary, in the in vitro system, DHEA caused a 1,5 - 4 fold induction of CYP2C9, GSTA1, DHEA-ST, 17ßHSD type II, CAR, PXR, RXR? and PPAR? mRNA expression, and a significant inhibition of CYP1A1, CYP2E1 and UGT2B17. The incubation with ADD, instead, evidenced only a significant inhibition of CYP1A1 expression. The different behavior observed in the in vivo and in vitro systems could be probably due to the dose applied and to the fact that the in vitro system represents a semplification of the organism in toto and that mimics with a lot of difficulties the pharmaco- and toxicological processes that normally happen in vivo (Freshney, 2001). Nonetheless, in vitro alternative methods are valid systems because they are rapid, riproducible and could substitute field experiments, that necessitate many animals. As a whole, other experiences, to standardize the protocol of hepatocytes isolation, are requested in order to reduce the inter-culture variability observed.

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Tipo di EPrint:Tesi di dottorato
Relatore:Dacasto, Mauro
Dottorato (corsi e scuole):Ciclo 20 > Scuole per il 20simo ciclo > SCIENZE VETERINARIE > SANITA' PUBBLICA VETERINARIA E PATOLOGIA COMPARATA
Data di deposito della tesi:31 Gennaio 2008
Anno di Pubblicazione:31 Gennaio 2008
Parole chiave (italiano / inglese):Citocromo P450; enzimi coniugativi; fattori di trascrizione; espressione genica; Real Time PCR; bovino; trattamenti illeciti; razza; epatociti in coltura primaria.
Settori scientifico-disciplinari MIUR:Area 07 - Scienze agrarie e veterinarie > VET/07 Farmacologia e tossicologia veterinaria
Struttura di riferimento:Dipartimenti > Dipartimento di Sanità pubblica, Patologia comparata ed Igiene veterinaria
Codice ID:252
Depositato il:24 Nov 2008
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