Effects of illicit drug treatments upon the cattle liver cytochrome P4503A (CYP3A) dependent hydroxylation of testosterone and protein expression.

Despite 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.