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In cattle, phenobarbital (PB) upregulates target drug-metabolizing enzyme (DME) mRNA levels. However, few data about PB's post-transcriptional effects are actually available. This work provides the first, and an almost complete, characterization of PB-dependent changes in DME catalytic activities in bovine liver using common probe substrates and confirmatory immunoblotting investigations. As expected, PB increased the total cytochrome P450 (CYP) content and the extent of metyrapone binding; moreover, an augmentation of protein amounts and related enzyme activities was observed for known PB targets such as CYP2B, 2C, and 3A, but also CYP2E1. However, contradictory results were obtained for CYP1A, while a decreased catalytic activity was observed for flavin-containing monooxygenases 1 and 3. The barbiturate had no effect on the chosen hydrolytic and conjugative DMEs. For the first time, we also measured the 26S proteasome activity, and the increase observed in PB-treated cattle would suggest this post-translational event might contribute to cattle DME regulation. Overall, this study increased the knowledge of cattle hepatic drug metabolism, and further confirmed the presence of species differences in DME expression and activity between cattle, humans, and rodents. This reinforced the need for an extensive characterization and understanding of comparative molecular mechanisms involved in expression, regulation, and function of DMEs. [ABSTRACT FROM AUTHOR]

Intestinal absorption of dietary fat is a complex process mediated by enterocytes leading to lipid assembly and secretion of circulating lipoproteins as chylomicrons, vLDL and intestinal HDL (iHDL). Understanding lipid digestion is of importance knowing the correlation between excessive fat absorption and atherosclerosis. By using time-of-flight secondary ion mass spectrometry (TOF-SIMS), we illustrated a spatio-temporal localization of fat in mice duodenum, at different times of digestion after a lipid gavage, for the first time. Fatty acids progressively increased in enterocytes as well as taurocholic acid, secreted by bile and engaged in the entero-hepatic re-absorption cycle. Cytosolic lipid droplets (CLD) from enterocytes were originally purified separating chylomicron-like, intermediate droplets and smaller HDL-like. A lipidomic quantification revealed their contents in triglycerides, free and esterified cholesterol, phosphatidylcholine, sphingomyelin and ceramides but also in free fatty acids, mono- and di-acylglycerols. An acyl-transferase activity was identified and the enzyme monoacylglycerol acyl transferase 2 (MGAT2) was immunodetected in all CLD. The largest droplets was also shown to contain the microsomal triglyceride transfer protein (MTTP), the acyl-coenzyme A-cholesterol acyltransferases (ACAT) 1 and 2, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL). This highlights the fact that during the digestion of fats, enterocyte CLD contain some enzymes involved in the different stages of the metabolism of diet fatty acids and cholesterol, in anticipation of the crucial work of endoplasmic reticulum in the process. The data further underlines the dual role of chylomicrons and iHDL in fat digestion which should help to efficiently complement lipid-lowering therapy. [ABSTRACT FROM AUTHOR]

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 6β-hydroxylase, −44.60% and −18.07%; testosterone 2β-hydroxylase, −43.95% and −11.69%); by contrast, an increase (about 2-fold) of the urinary 6β-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 6β-hydroxycortisol:cortisol ratio is not useful to monitor in vivo CYP3A activity in DEX-treated individuals. [Copyright &y& Elsevier]

Abstract: The effects of the administration of a combination of 17β-estradiol (10mg i.m. for three times at 17 days intervals), dexamethasone (4mg/day for 6 days and 5mg/day for further 6 days, dissolved in milk), and clenbuterol (20μg/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. [Copyright &y& Elsevier]

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, β-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 17β-oestradiol (10mg, 3im injections at 17 days intervals), clenbuterol (20μgkg−1, per os for 40 days) and dexamethasone (4mg 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 T0 and after 15 (T1), 34 (T2), 48 (T3) days as well as the day before slaughtering (T4). Immune endpoints considered were the thymus weight, the serum immunoglobulin G (IgG) and M (IgM) levels, the lymphocyte proliferation assay and the lymphocyte interleukins 1β and 8, tumour necrosis factor α and interferon γ (IFN-γ) 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 T1, whereas in the second part of the study increasing levels (P <0.05 at T2 and T4 for IgM and IgG, respectively) were recorded; (c) an overall reduction (P <0.001, P <0.05) of lymphocyte proliferation rate at T1; in phytohaemagglutinin-stimulated cells, such a decrease was delayed up to T2 (P <0.05); (d) a reduction (P <0.05) in IFN-γ mRNA levels at T1 and T2. 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. [Copyright &y& Elsevier]

Abstract: Low doses of the synthetic glucocorticoid dexamethasone (DEX) are often illegally used, alone or in association with steroids and β-agonists, to improve meat performances in cattle. As it is known that oestrogens and β-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. [Copyright &y& Elsevier]

Background: Intestinal absorption of dietary lipids involves their hydrolysis in the lumen of proximal intestine as well as uptake, intracellular transport and re-assembly of hydrolyzed lipids in enterocytes, leading to the formation and secretion of the lipoproteins chylomicrons and HDL. In this study, we examined the potential involvement of cytosolic lipid droplets (CLD) whose function in the process of lipid absorption is poorly understood. Methods: Intestinal lipid absorption was studied in mouse after gavage. Three populations of CLD were purified by density ultracentrifugations, as well as the brush border membranes, which were analyzed by western-blots. Immunofluorescent localization of membranes transporters or metabolic enzymes, as well as kinetics of CLD production, were also studied in intestine or Caco-2 cells. Results: We isolated three populations of CLD (ranging from 15 to 1000 nm) which showed differential expression of the major lipid transporters scavenger receptor BI (SR-BI), cluster of differentiation 36 (CD-36), Niemann Pick C-like 1 (NPC1L1), and the ATP-binding cassette transporters ABCG5/G8 but also caveolin 2 and fatty acid binding proteins. The enzyme monoacylglycerol acyltransferase 2 (MGAT2) was identified in the brush border membrane (BBM) in addition to the endoplasmic reticulum, suggesting local synthesis of triglycerides and CLD at both places. Conclusions: We show a very fast production of CLD by enterocytes associated with a transfer of apical constituents as lipid transporters. Our findings suggest that following their uptake by enterocytes, lipids can be partially metabolized at the BBM and packaged into CLD for their transportation to the ER. [ABSTRACT FROM AUTHOR]