[Show abstract][Hide abstract] ABSTRACT: The intestinal mucosa plays a capital role in dictating the bioavailability of a large array of orally ingested drugs and toxicants. The activity and the expression of several xenobiotic metabolizing enzymes were measured in subcellular fractions from the duodenal mucosa of male veal calves and beef cattle displaying a functional rumen but differing in both age (about 8 months vs. 18 to 24 months) and dietary regimens (i.e., milk replacer plus hay and straw vs. corn and concentrated meal). Intestinal microsomes showed cytochrome P450 (CYP) 2B, 2C- and 3A-mediated activities and the presence of the corresponding immunorelated proteins, but no proof of CYP1A expression and/or functions could be provided. Intestinal microsomes were also active in performing reactions typically mediated by carboxylesterases (indophenylacetate hydrolysis), flavin-containing monooxygenases (methimazole S-oxidation), and uridindiphosphoglucuronyltransferases (1-naphthol glucuronidation), respectively. Cytosolic fractions displayed the glutathione S-transferase (GST)-dependent conjugation of 1-chloro-2,4-dinitrobenzene; besides, the GST-mediated conjugation of ethacrinic acid (GSTpi) or cumene hydroperoxide (GSTalpha) was matched by the presence of the corresponding immunorelated proteins. Conversely, despite the lack of measurable activity with 3,4-dichloronitrobenzene, a protein cross reacting with anti-rat GSTmu antibodies could be clearly detected. Although, as detected by densitometry, CYPs and GST isoenzymes tended to be more expressed in beef cattle than in veal calf preparations, there was a general poor correlation with the rate of the in vitro metabolism of the selected diagnostic probes.
Journal of Veterinary Pharmacology and Therapeutics 06/2010; 33(3):295-303. DOI:10.1111/j.1365-2885.2009.01137.x · 1.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dexamethasone (DEX) exerts its known anti-inflammatory and immunosuppressant activities through the interaction with the glucocorticoid receptor (GR). In human liver, DEX is metabolized by cytochrome P450 3A (CYP3A); moreover, it is among those xenobiotics which induce CYP3A itself. The transcriptional regulation of CYP3A involves GR and nuclear receptors (NRs). In cattle, DEX is used at low dosages as a growth promoter; besides, CYP3A is expressed in the liver. In the present study, the effects of two illicit DEX protocols upon liver CYP3A were investigated in the veal calf. Dexamethasone, administered per os (DOS) or injected intramuscularly (DIM) at growth promoting purposes, increased GR mRNA (+25.62% and +73.02% of CTRL for DOS and DIM, respectively), while tyrosine aminotransferase (TAT) and NRs gene expression profiles were unaffected; decreased CYP3A mRNA (-20.64% and -16.07% with Q RT-PCR; -30.55% and -34.31% with Northern blotting); at the post-translational level, decreased TAT activity (-19.84% and 44.34%), CYP3A apoprotein (-27.65% and -42.85%) and CYP3A-dependent enzyme activities (erythromycin N-demethylase, -78.89% and -23.87%; ethylmorphine N-demethylase, -44.26% and -28.37%; testosterone 6beta-hydroxylase, -44.60% and -18.07%; testosterone 2beta-hydroxylase, -43.95% and -11.69%); by contrast, an increase (about 2-fold) of the urinary 6beta-hydroxycortisol:cortisol ratio was observed in vivo. In summary, DEX modulates cattle liver CYP3A at pre- and post-translational level. Species-differences in GR-NRs-CYP3A regulation and in their response to differing DEX dosages might justify present results. Furthermore, the urinary 6beta-hydroxycortisol:cortisol ratio is not useful to monitor in vivo CYP3A activity in DEX-treated individuals.
[Show abstract][Hide abstract] ABSTRACT: The effects of the administration of a combination of 17beta-estradiol (10mg i.m. for three times at 17 days intervals), dexamethasone (4 mg/day for 6 days and 5mg/day for further 6 days, dissolved in milk), and clenbuterol (20 microg/kg b.w./day, dissolved in milk, for the last 40 days before slaughtering) for growth-promoting (GP) purposes on liver drug metabolising capacity were studied in crossbred Friesian male calves. Compared to controls, liver preparations from GP-treated calves showed an overall reduction in the extent of the in vitro ability to metabolize testosterone and a number of substrates, most notably those associated with CYP 2C or CYP 3A, which also displayed a reduced expression on western blotting. By contrast, the tested hydrolytic and conjugative pathways were not significantly affected. As measured by northern blot, the lack of significant differences in CYP mRNA abundance point to a post-transcriptional effect of the GP combination. The remarkable involvement of the affected hepatic CYPs in the biotransformation of both steroid hormones and a large array of commonly used drugs may result in the further accumulation of undesirable residues in meat and offals of illegally treated calves.
Food and Chemical Toxicology 09/2008; 46(8):2849-55. DOI:10.1016/j.fct.2008.05.018 · 2.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cattle represent an important source of animal-derived food-products; nonetheless, our knowledge about the expression of drug-metabolizing enzymes (DMEs) in present and other food-producing animals still remains superficial, despite the obvious toxicological consequences. Breed represents an internal factor that modulates DME expression and catalytic activity. In the present work, the effect of breed upon relevant phase I and phase II DMEs was investigated at the pretranscriptional and post-translational levels in male Charolais (CH), Piedmontese (PM) and Blonde d'Aquitaine (BA) cattle. Because specific substrates for cattle have not yet been identified, the breed effect upon specific cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), or glutathione S-transferase (GST) DMEs, in terms of catalytic activity, was determined by using human marker substrates. Among P450s, benzphetamine N-demethylase, 16beta-, 6beta-, and 2beta-testosterone hydroxylase, aniline and p-nitrophenol hydroxylase, and alpha-naphthol and p-nitrophenol UGT activities were significantly higher in CH; in contrast, lower levels of CYP1A1-, CYP1A2-, CYP2B6-, CYP2C9-, CYP2C18-, CYP3A4-, and UGT1A1-like mRNAs were noticed, with CH < PM < or = BA as a trend. CYP2B and CYP3A mRNA results were confirmed with immunoblotting, too. As regards conjugative DMEs, UGT1A6-like mRNA levels were consistent with respective catalytic activities. Both 1-chloro-2,4-dinitrobenzene and 3,4-dichloronitrobenzene GST activities were higher in BA, and these results agreed with GSTA1-, GSTM1-, and GSTP1-like mRNA amounts. Correlation analysis between catalytic activities and mRNAs showed either significant or uneven results, depending on the substrate. These findings confirm previous data obtained in laboratory species; however, further studies are required to ascribe this behavior to pretranscriptional or post-translational phenomena.
Drug metabolism and disposition: the biological fate of chemicals 05/2008; 36(5):885-93. DOI:10.1124/dmd.107.019042 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: At the European Union level, the use of growth promoters (GPs) in cattle and other food-producing species is forbidden; nonetheless, the illicit use of anabolic hormones, beta-agonists and corticosteroids, often administered in cocktails at lower concentrations to overcome control procedures, is still of public concern. The immune system (IS) is a multicomponent system that provide a coordinated response toward infectious diseases, not self-neoplasms and xenobiotics; in this respect, some GPs have been proved able to cause both morphological alterations in lymphoid organs and a modulating effect upon some immunological parameters. Therefore, in the present study the effects of an illicit cocktail upon the cattle IS functions were investigated by using some common endpoints adopted for the IS testing in humans. Twelve cross-bred male veal calves were divided in two experimental groups (n=6); the first group was administered a cocktail of 17beta-oestradiol (10 mg, 3 im injections at 17 days intervals), clenbuterol (20 microg kg(-1), per os for 40 days) and dexamethasone (4 mg per os for 6 days and, then, 5mg for further 6 days) for a total of 55 days. The second one was used as control. Blood sampling were taken at T(0) and after 15 (T(1)), 34 (T(2)), 48 (T(3)) days as well as the day before slaughtering (T(4)). Immune endpoints considered were the thymus weight, the serum immunoglobulin G (IgG) and M (IgM) levels, the lymphocyte proliferation assay and the lymphocyte interleukins 1beta and 8, tumour necrosis factor alpha and interferon gamma (IFN-gamma) gene expression levels. The administration of the illicit cocktail resulted in: (a) a reduction (P<0.01) of both the absolute and relative thymus weight; (b) a decrease (P<0.05) of both IgG and IgM serum levels at T(1), whereas in the second part of the study increasing levels (P<0.05 at T(2) and T(4) for IgM and IgG, respectively) were recorded; (c) an overall reduction (P<0.001, P<0.05) of lymphocyte proliferation rate at T(1); in phytohaemagglutinin-stimulated cells, such a decrease was delayed up to T(2) (P<0.05); (d) a reduction (P<0.05) in IFN-gamma mRNA levels at T(1) and T(2). Taken together, present data suggest that GPs, even given in cocktails at sub-therapeutic dosages, can modulate the cattle IS, thereby hampering itself to exert its physiological role in defence mechanisms. Further studies are required to confirm and investigate these results.
[Show abstract][Hide abstract] ABSTRACT: Low doses of the synthetic glucocorticoid dexamethasone (DEX) are often illegally used, alone or in association with steroids and beta-agonists, to improve meat performances in cattle. As it is known that oestrogens and beta-agonists may generate reactive oxygen species (ROS) and induce oxidative stress, the effects of illicit DEX protocols on the antioxidant status and oxidative stress parameters were measured in veal calves. Ten cross-bred male veal calves were given DEX (0.4mg/day administered per os, for 23days or 2mg pro capite, injected intramuscularly on days 14 and 21 after the beginning of the oral DEX administration). Five further animals were used as controls. Blood samples were withdrawn before (T(0)), and 4 (T(1)), 10 (T(2)), 14 (T(3)), 21 (T(4)) and 28 (T(5)) days. Antioxidant enzyme activities (AOEs), the serum antioxidant capacity (SAC) and ROS were measured in sera. Calves orally treated showed a significant increase of both glutathione peroxidase isoforms (P<0.05) and SAC (P<0.05), too. Antioxidant enzymes have already been used as biomarkers (BMs) of response, measured in target or in surrogate tissues. Our results suggest glutathione peroxidase and SAC as possible BMs of illicit oral low-dose administration of DEX in cattle.
Toxicology in Vitro 04/2007; 21(2):277-83. DOI:10.1016/j.tiv.2006.09.001 · 2.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glucocorticoids are often illegally used in association with anabolic steroids as growth promoters in veal calves and beef production. An experimental administration of dexamethasone was carried out in veal calves in order to assess the role of low doses of exogenous glucocorticoids on induction of thymus atrophy and on the immune response. Three groups of five veal calves each were included in this study: group D was administered 0.4 mg/day of dexamethasone-21-phosphate per os for 25 days; group V was administered 2 mg of dexamethasone-21-isonicotinate i.m. at days 14 and 21, and group K served as control. At slaughter, the weight of the thymus was severely reduced in group D and in group V, compared with control animals. Lesions included severe lymphoid depletion and hyperplasia of adipose tissue. In situ evaluation of apoptosis in thymus, showed a reduction of the percentage of positive nuclear areas of animals belonging to group V in comparison with control animals. An overall decrease of lymphocyte proliferative response was detected after treatment with short acting dexamethasone, while antibody response was not affected by treatments.
Journal of Veterinary Medicine Series A 06/2005; 52(4):202-8. DOI:10.1111/j.1439-0442.2005.00714.x · 0.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nuclear hormone receptors (NR) are important transcriptional regulators of numerous genes involved in diverse pathophysiological and therapeutic functions. Following ligand activation, class II NR share the ability to heterodimerize with the retinoid X receptor (RXR). It is established that RXR activators, rexinoids, transactivate several peroxisome proliferator-activated receptor alpha (PPARalpha) target genes in a PPARalpha-dependent manner. We hypothesized that, once activated, RXR might signal through quiescent NR other than PPARalpha, in an organ-specific manner. To study this putative phenomenon in vivo, we developed an array of 120 genes relevant to the class II NR field. The genes were selected using both published data and high-density screenings performed on RXR or PPARalpha agonist-treated mice. Wild-type C57BL/6J and PPARalpha-deficient mice were treated with fenofibrate (PPARalpha activator) or LGD1069 (RXR activator). Using our customized array, we studied the hepatic, cardiac, and renal expression of this panel of 120 genes and compared them in both murine genotypes. The results obtained from this study confirmed the ability of an RXR agonist to modulate PPARalpha-restricted target genes in the liver and the kidney. Furthermore, we show that various organ-specific regulations occurring in both genotypes (PPARalpha +/+ or -/-) are highly indicative of the ability of RXR to recruit other class II NR pathways. Further development of this molecular tool may lead to a better understanding of the permissiveness of class II nuclear receptor dimers in vivo.