Intestinal biotransformation in sheep. Comparative assessment of phase 1 and 2 metabolic activities in the mucosa of duodenum, jejunum and ileum

VIRKEL G.; MATÉ L.; LIFSCHITZ A.; SALLOVITZ J.; BALLENT M.; LANUSSE C

Leipzig (Alemania)

Congreso; 11 th Congress of the European Association of Veterinary Pharmacology and Therapeutics (EAVPT); 2009

European Assocition of Veterinary Pharmacology and Therapeutics

The intestinal mucosa constitutes an absorptive barrier in the uptake of toxic compounds or orally administered drugs. In addition, it also has the ability to metabolise a great number of xenobiotics by numerous pathways involving both phase 1 and phase 2 reactions [1]. Most of the published works on this subject have been carried out in laboratory animals and man but little is known concerning the metabolism of xenobiotics in the intestinal mucosa in ruminant species. Previously, species differences in phase 1 and 2 enzymatic activities were observed in microsomal and cytosolic preparations from the ileal mucosa of sheep and cattle [2]. Both benzphetamine and erythromycin N-demethylase activities were measured in microsomes obtained from different segments of sheep intestinal mucosa and the presence of P450 2B and 3A immunoreacting proteins was also demonstrated in this tissue [3]. Glucuronosyl transferase (UGT), glutathione S-transferase (GST) and N-acetyl transferase activities were also characterized along the GI tract of female Lacaune sheep [4]. The aim of this research was to evaluate the activities of different phase 1 and phase 2 xenobiotic metabolizing enzymes in cytosolic and microsomal fractions obtained from the mucosa of different segments of sheep small intestine. The same enzymatic activities measured in liver subcellular fractions were used as reference values. MATERIALS AND METHODS Samples of liver tissue (caudate lobe) and segments of duodenum, jejunum and ileum were obtained after sacrifice of healthy Corriedale x Merino cross-breed rams (n=8). The intestinal mucosa was obtained by scraping using a microscope glass slide. Microsomal and cytosolic fractions were obtained following previously described procedures [5,6]. Subcellular fractions were used to measure the rate of the in vitro metabolism of several phase 1 and phase 2 substrates that in humans and other laboratory or livestock species are believed to be markers for the expression of oxidative, reductive, hydrolytic and conjugative enzymes [5,7,8]. Enzymatic activities were also measured in liver subcellular fractions and used as reference values (equal to 100 % of a given metabolic assay). Metabolic activities were compared using the Kruskal-Wallis test (nonparametric ANOVA). A value of P<0.05 was considered significant. RESULTS AND DISCUSSION No measurable microsomal ethoxyresorufin O-deethylase and ethoxycoumarin O-deethylase were observed in microsomes from sheep small intestinal mucosa. Table 1 shows mean (±SD) values obtained for the oxidative (P450 and FMO-mediated), reductive, hydrolytic and conjugative enzymatic activities quantified. Cytochrome P450-mediated N-demethylase activities did not decrease nor increase along the small intestine. Conversely, flavin-monooxygenase (FMO) activity increased (2.6-fold) from duodenum to ileum. Carboxyl esterase activity towards p-nitrophenyl acetate (p-NPA) was 43 % lower in the ileum compared to duodenum. Carbonyl reductase (CBR) and GST activities were similar in all intestinal segments, whilst UGT avtivity decreased from duodenum to ileum. The small intestinal mucosa may play a critical defensive role due to the detoxification of toxic compounds prior to absorption. In addition, metabolic reactions taking place in this tissue may contribute to the pre-systemic metabolism of orally administered drugs. The results presented in this work are a further contribution to the knowledge of xenobiotic metabolizing enzymes in the small intestine of ruminant species.