Introduction. Growth promoters (GPs), although banned within the European Union (Directive 88/146/EEC) are still used in cattle, alone and/or in combinations, to increase growth and improve the carcass quality (1). Among these ones, relevant importance harvest the anabolic agent boldenone (BOL) and its precursor, boldione (ADD). One of the major subject of debate on BOL and ADD concern their “endogenous” nature, the possible conversion, by some microorganisms of dietary phytosterols, as -sitosterol, into ADD and then BOL (2). Therefore, a study aiming to evaluate the effect of different sterols-containing diets a well as ADD and BOL administration on the cytochrome P450-dependent liver drug metabolising enzymes (P450-DMEs) activities, was undertaken in the veal calf. Materials and Methods. The experiment involved, according to a 2x2 design, 28 German Brown male calves (10 days old, about 68.7±0.14 Kg bw). Calves were kept in separate boxes for 3 months and fed a commercial milk replacer. At the end of this period, the diet was changed: fourteen animals received a milk replacer added with an higher percentage of plant sterols (VS) whereas the other 14 were given the milk with sterols of animal origin (AS). At the age of about 5 months, 7 calves out of 14 for each group (VST and AST) received daily, per os and pro capite, a combination of BOL and ADD (0,9 mg and 0,1 mg, respectively), for 40 days. The remaining 7 animals of each group (VSK and ASK) were used as control and administered only milk replacers. All the animals were slaughtered the day after the last treatment (245.3±15.52 Kg bw). Main P450-DMEs activities and protein expression levels (immunoblotting) were measured, in subcellular microsomal fractions obtained from the liver caudate lobe, according to previously published methods (3-5). Statistical analyses of paired (VSK vs VST, ASK vs AST, VSK vs ASK and VST vs AST) and grouped data were performed by using the Mann-Whitney U-Test or the analysis of variance (ANOVA), followed by a post-test (Tukey-Kramer multiple comparisons test). Results. Enzyme activities, expressed as mean values ± standard deviation (SD) are reported in Table 1. A slight decrease of P450-DMEs activities was observed in VSK and VST calves, fed the milk replacer containing vegetal sterols, compared to ASK and AST ones. No statistically significant differences were ever noticed between paired or grouped data, even at the protein expression level. Discussion and Conclusion. In past years, several studies aiming to ascertain the possible effects of GPs upon P450-DMEs have been executed; as a whole, these studies gave contradictory results (6-8). Several factors might partly explain such an evidence: the type of drug used and its formulation (i.e. anabolics, corticosteroids, drugs used alone or in combination), the breeding type (veal calves, beef cattle), nutritional factors (low iron-diet, milk replacer, unifeed), modulation of animal welfare (i.e. blood sampling, weighing). In the present study, BOL and ADD were unable to modulate the P450-DMEs activities and protein expression levels, even in presence of diets containing animal or plant sterols. Recently some P450-DMEs activities have been currently used as biomarkers of exposure in biological monitoring (9); to the best of our previous and present data, these enzymes, if strictly limited to their catalytic activity or protein expression level, cannot be used as a screening test for illicit drug treatments in cattle.

Influence of diets containing animal or plant sterols and a boldenone/boldione combination on liver drug metabolism in veal calves.

MERLANTI, ROBERTA;CAPOLONGO, FRANCESCA;POPPI, LISA;DACASTO, MAURO;MONTESISSA, CLARA
2006

Abstract

Introduction. Growth promoters (GPs), although banned within the European Union (Directive 88/146/EEC) are still used in cattle, alone and/or in combinations, to increase growth and improve the carcass quality (1). Among these ones, relevant importance harvest the anabolic agent boldenone (BOL) and its precursor, boldione (ADD). One of the major subject of debate on BOL and ADD concern their “endogenous” nature, the possible conversion, by some microorganisms of dietary phytosterols, as -sitosterol, into ADD and then BOL (2). Therefore, a study aiming to evaluate the effect of different sterols-containing diets a well as ADD and BOL administration on the cytochrome P450-dependent liver drug metabolising enzymes (P450-DMEs) activities, was undertaken in the veal calf. Materials and Methods. The experiment involved, according to a 2x2 design, 28 German Brown male calves (10 days old, about 68.7±0.14 Kg bw). Calves were kept in separate boxes for 3 months and fed a commercial milk replacer. At the end of this period, the diet was changed: fourteen animals received a milk replacer added with an higher percentage of plant sterols (VS) whereas the other 14 were given the milk with sterols of animal origin (AS). At the age of about 5 months, 7 calves out of 14 for each group (VST and AST) received daily, per os and pro capite, a combination of BOL and ADD (0,9 mg and 0,1 mg, respectively), for 40 days. The remaining 7 animals of each group (VSK and ASK) were used as control and administered only milk replacers. All the animals were slaughtered the day after the last treatment (245.3±15.52 Kg bw). Main P450-DMEs activities and protein expression levels (immunoblotting) were measured, in subcellular microsomal fractions obtained from the liver caudate lobe, according to previously published methods (3-5). Statistical analyses of paired (VSK vs VST, ASK vs AST, VSK vs ASK and VST vs AST) and grouped data were performed by using the Mann-Whitney U-Test or the analysis of variance (ANOVA), followed by a post-test (Tukey-Kramer multiple comparisons test). Results. Enzyme activities, expressed as mean values ± standard deviation (SD) are reported in Table 1. A slight decrease of P450-DMEs activities was observed in VSK and VST calves, fed the milk replacer containing vegetal sterols, compared to ASK and AST ones. No statistically significant differences were ever noticed between paired or grouped data, even at the protein expression level. Discussion and Conclusion. In past years, several studies aiming to ascertain the possible effects of GPs upon P450-DMEs have been executed; as a whole, these studies gave contradictory results (6-8). Several factors might partly explain such an evidence: the type of drug used and its formulation (i.e. anabolics, corticosteroids, drugs used alone or in combination), the breeding type (veal calves, beef cattle), nutritional factors (low iron-diet, milk replacer, unifeed), modulation of animal welfare (i.e. blood sampling, weighing). In the present study, BOL and ADD were unable to modulate the P450-DMEs activities and protein expression levels, even in presence of diets containing animal or plant sterols. Recently some P450-DMEs activities have been currently used as biomarkers of exposure in biological monitoring (9); to the best of our previous and present data, these enzymes, if strictly limited to their catalytic activity or protein expression level, cannot be used as a screening test for illicit drug treatments in cattle.
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