Characterization of xenobiotic metabolizing enzymes in bovine small intestinal mucosa

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.

Cytochrome P450 3A expression and function in liver and intestinal mucosa from dexamethasone‐treated sheep

“…Microsomal and cytosolic fractions from both hepatic tissue and intestinal mucosa were isolated by differential ultracentrifugation as previously described (Virkel et al. , 2010).…”

Constitutive expression of the AHR signaling pathway in a bovine mammary epithelial cell line and modulation by dioxin-like PCB and other AHR ligands

“…After blocking of non-specific binding sites with 10% BSA in TBS, the membranes have been incubated for 2 h at room temperature with primary antibodies against rabbit CYP1A1/1A2 (Oxford Biomedical Research, Oxford, MI). The specificity of the CYP1A antibody against the bovine protein has been previously ascertained (Virkel et al, 2010). A primary antibody against b-tubulin (Santa Cruz Biotechnology, CA) was used as loading control.…”

Modulating nasal mucosal permeation using metabolic saturation and enzyme inhibition techniques

“…An additional limitation to the reproducible use of cell cultures for nasal transport studies is the lack of an immortalized nasal cell line that simulates the nasal epithelial and submucosal tissue interactions. The use of bovine tissues for studying the enzymatic activity of various organs like the liver, lungs, intestinal mucosa, kidneys, coronary arteries and tongue has become increasingly popular in recent years . This species has also been shown to be well‐suited for investigations of nasal permeation and metabolism .…”

“…The use of bovine tissues for studying the enzymatic activity of various organs like the liver, lungs, intestinal mucosa, kidneys, coronary arteries and tongue has become increasingly popular in recent years. [24][25][26][27] This species has also been shown to be well-suited for investigations of nasal permeation and metabolism. [28] Using real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and enzyme immunohistochemistry, we have previously demonstrated that CYP450 isoforms and corresponding proteins are expressed in the bovine nasal mucosa, and the enzymes are localized both in the epithelial as well as submucosal regions.…”