Intestinal Slices Research Articles

P024 Regional differences in effect of TGF-beta1 and PDGF on the early onset of intestinal fibrosis

Background: Intestinal fibrosis (IF) is one of the major complications in inflammatory bowel disease patients. IF can cause narrowing of the intestinal lumen, which may lead to stricture formation. The mechanism of IF is still unknown and adequate models are lacking. By using precision-cut intestinal slice (PCIS) from different regions of the bowel, we studied the early onset of fibrosis in mouse jejunum, ileum and colon PCIS, in the presence of transforming growth factor (TGF)-beta1 and platelet-derived growth factor (PDGF). Methods: Mouse jejunum, ileum and colon were excised and prepared as a segment embedded in agarose. PCIS (estimated 300-400 μm) was prepared and incubated up to 48 hr with or without the presence of TGF-beta1 and PDGF. ATP content of the PCIS was used to assess the general viability. The gene expression of different fibrosis markers including Pro-Collagen 1 A1 (COL1A1), heat shock protein 47 (HSP47), alpha-smooth muscle actin (SMA), connective tissue growth factor (CTGF), synaptophysin (SYN) and fibronectin (FN2) were determined. Results: Mouse PCIS from different segments were viable up to 48 hr. After 48 hr of incubation, HSP47 and FN2 gene expression were significantly up-regulated, compared to PCIS directly after slicing, in jejunum (3.6 and 4.9 fold, respectively) and in ileum (4.9 and 5.5 fold, respectively). When incubated with 5 ng/mL TGF-beta1, in jejunum PCIS, COL1A1, HSP47, CTGF and FN2 were significantly up-regulated compared to 48 hr control. In ileum PCIS the gene expression of HSP47 (1.9 fold) and FN2 (3.9 fold) were also significantly increased. In the presence of 50 ng/mL PDGF, only in ileum PCIS, CTGF (1.4 fold) and SYN (1.9 fold) were significantly increased compared to 48 hr control. Interestingly, in PCIS from the colon, 5 ng/ml TGF-beta1 did not affect the gene expression of the fibrosis markers. However, HSP47 (1.4 fold) and FN2 (1.7 fold) were significantly increased when colon PCIS were incubated with 50 ng/mL PDGF. Conclusions: TGF-beta1, but not PDGF, was able to induce HSP47 and FN2 in mouse jejunum and ileum PCIS. This is in contrast to the result in colon PCIS, where only PDGF was able to induce these fibrosis markers. Moreover, PDGF increased CTGF and SYN only in ileum PCIS. These results indicate differences in the effect of TGF-beta1 and PDGF on the early onset of fibrosis in different regions of the intestine.

P025 Reduced Butyricicoccus pullicaecorum levels in mucosa of UC patients correlate with aberrant CLDN1 expression

Background: Intestinal fibrosis (IF) is one of the major complications in inflammatory bowel disease patients. IF can cause narrowing of the intestinal lumen, which may lead to stricture formation. The mechanism of IF is still unknown and adequate models are lacking. By using precision-cut intestinal slice (PCIS) from different regions of the bowel, we studied the early onset of fibrosis in mouse jejunum, ileum and colon PCIS, in the presence of transforming growth factor (TGF)-beta1 and platelet-derived growth factor (PDGF). Methods: Mouse jejunum, ileum and colon were excised and prepared as a segment embedded in agarose. PCIS (estimated 300 400mm) was prepared and incubated up to 48 hr with or without the presence of TGF-beta1 and PDGF. ATP content of the PCIS was used to assess the general viability. The gene expression of different fibrosis markers including Pro-Collagen 1 A1 (COL1A1), heat shock protein 47 (HSP47), alpha-smooth muscle actin (SMA), connective tissue growth factor (CTGF), synaptophysin (SYN) and fibronectin (FN2) were determined. Results: Mouse PCIS from different segments were viable up to 48 hr. After 48 hr of incubation, HSP47 and FN2 gene expression were significantly up-regulated, compared to PCIS directly after slicing, in jejunum (3.6 and 4.9 fold, respectively) and in ileum (4.9 and 5.5 fold, respectively). When incubated with 5 ng/mL TGF-beta1, in jejunum PCIS, COL1A1, HSP47, CTGF and FN2 were significantly up-regulated compared to 48 hr control. In ileum PCIS the gene expression of HSP47 (1.9 fold) and FN2 (3.9 fold) were also significantly increased. In the presence of 50 ng/mL PDGF, only in ileum PCIS, CTGF (1.4 fold) and SYN (1.9 fold) were significantly increased compared to 48 hr control. Interestingly, in PCIS from the colon, 5 ng/ml TGF-beta1 did not affect the gene expression of the fibrosis markers. However, HSP47 (1.4 fold) and FN2 (1.7 fold) were significantly increased when colon PCIS were incubated with 50 ng/mL PDGF. Conclusions: TGF-beta1, but not PDGF, was able to induce HSP47 and FN2 in mouse jejunum and ileum PCIS. This is in contrast to the result in colon PCIS, where only PDGF was able to induce these fibrosis markers. Moreover, PDGF increased CTGF and SYN only in ileum PCIS. These results indicate differences in the effect of TGF-beta1 and PDGF on the early onset of fibrosis in different regions of the intestine.

Precision-cut intestinal slices as an in vitro model to predict NSAID induced intestinal toxicity

Non-steroidal anti-inflammatory drugs (NSAIDs) are associated with high prevalence of gastro-intestinal side-effects. In vivo studies suggest that uncoupling of oxidative phosphorylation is an important cause of the toxicity and that the toxicity is aggravated by enterohepatic circulation. Precision-cut intestinal slices (PCIS) are a newly established in vitro model with intact architecture of the intestine, preserved viability and metabolism during incubation up to 24 h. In this study, this model is used to predict NSAID-induced intestinal toxicity. PCIS from rat jejunum were incubated for 5 h with a concentration range of 5 NSAIDs: diflunisal (0.05-1 mM), indomethacin (0.05-1 mM), diclofenac (0.05-1 mM), naproxen (0.1-5mM) and aspirin (0.1-10 mM). Toxicity was assessed by the ATP content. In vitro TC50 of these 5 compounds was calculated and compared with in vivo TC50 and the mitochondrial toxicity data as reported in the literature. Based on TC50 values, toxicity ranking in the present study appeared to be: aspirin (>10mM)> naproxen (2.96mM)> diclofenac (0.29mM)> indomethacin (0.29mM)> diflunisal (0.18 mM). This is very well in line with the reported toxicity ranking found with the isolated mitochondria. When compared with the in vivo TC50, only one exception of diflunisal was found, which appeared highly toxic in vitro whereas in vivo it's one of the least toxic compound. This discrepancy could not yet be explained by in vivo toxicokinetic factors (absorbtion, enterohepatic circulation, metabolism, excretion). In conclusion, both intrinsic (mitochondrial) toxicity as well as in vivo toxicity ranking are well predicted by the PCIS model.

Nanoparticle Formulation of a Poorly Soluble Cannabinoid Receptor 1 Antagonist Improves Absorption by Rat and Human Intestine

The inclusion of nanoparticles dispersed in a hydrophilic matrix is one of the formulation strategies to improve the bioavailability of orally administered Biopharmaceutics Classification System (BCS) class II and IV drugs by increasing their dissolution rate in the intestine. To confirm that the increased dissolution rate results in increased bioavailability, in vitro and in vivo animal experiments are performed, however, translation to the human situation is hazardous. In this study, we used a range of in vitro and ex vivo methods, including methods applying human tissue, to predict the in vivo oral bioavailability of a model BCS class II CB-1 antagonist, formulated as a nanoparticle solid dispersion. The enhanced dissolution rate from the nanoparticle formulation resulted in an increased metabolite formation in both rat and human precision-cut intestinal slices, suggesting increased uptake and intracellular drug concentration in the enterocytes. In Ussing chamber experiments with human tissue, both the metabolite formation and apical efflux of the metabolite were increased for the nanoparticulate solid dispersion compared with a physical mixture, in line with the results in intestinal slices. The pharmacokinetics of the different formulations was studied in rats in vivo. The nanoparticle formulation indeed improved the absorption of the cannabinoid receptor 1 (CB-1) antagonist and the delivery into the brain compared with the physical mixture. In conclusion, the combined approach provides a valuable set of tools to investigate the effects of formulation on the absorption of poorly soluble compounds in human intestine and may provide relevant information on the oral bioavailability in humans early in the development process.

Mo1823 Involvement of MDR1 in Intestinal Epithelial Injury Caused by Acetylsalicylic Acid

Introduction Intestinal fibrosis (IF) is a major complication of chronic inflammation of the intestinal tissue in inflammatory bowel disease (IBD). IF can cause narrowing of the intestinal lumen, which may lead to stricture formation. Currently, adequate models are lacking to study the mechanism of intestinal fibrosis in IBD. Our aim is to develop an ex vivo model for IF by using mouse precision-cut intestinal slice (PCIS). In addition, regional differences in the onset of fibrosis in the bowel will be studied. In PCIS all cell types are present in their original tissue-matrix environment. As fibrosis is a multicellular phenomenon, PCIS can be used as a model to study the early onset of intestinal fibrosis. Methods Mouse jejunum, ileum and colon were excised and prepared as segments embedded in agarose. PCIS (estimated 300-400 μm) were prepared and incubated up to 48 hr. ATP content of the PCIS was used to assess the general viability. To study the early onset of fibrosis, the gene expression of different fibrosis markers: Pro-Collagen 1 A1 (COL1A1), Heat Shock Protein 47 (HSP47), alpha-Smooth Muscle Actin (SMA) and Fibronectin (FN1) was determined in PCIS after 48 hr of incubation. Results Mouse PCIS from the different regions of the bowel were viable up to 48 hr in culture. After 48 hr of incubation of PCIS, HSP47 and FN1 gene expression was significantly up-regulated, compared to PCIS directly after slicing, in jejunum (3.9 and 3.6 fold, respectively) and in ileum (4.5 and 4.9 fold, respectively). Gene expression of Col1A1 (0.7 fold) was only down-regulated in PCIS from the colon. SMA gene expression was down-regulated in PCIS from all the regions of the intestine. PCIS from jejunum cultured in the presence of 5ng/mL Transforming-Growth Factor (TGF)β1 for 48 hr, HSP47 (1.2 fold), FN1 (4.6 fold) and COL1A1 (2.0 fold) were significantly upregulated compared to control PCIS after 48 hr of incubation. In addition, in PCIS from Ileum incubated with 5ng/mL TGF-β1, the gene expression of HSP47 (1.9 fold) and FN1 (3.9 fold) was significantly increased. In PCIS from the colon cultured for 48 hr in the presence of 5ng/ml TGF-β1, the gene expression of fibrosis markers was not affected. Conclusion Mouse PCIS from different regions of the bowel can successfully be cultured for 48 hr. Fibrosis markers are induced by incubation up to 48 hr and by addition of TGFβ1 in jejunum and ileum PCIS, but not in colon PCIS. We are the first to show that regional differences in the onset of intestinal fibrosis can be measured using PCIS.

P067 Human and rodent ex vivo model for intestinal fibrosis in inflammatory bowel disease (IBD)

Background: One of the major complications in IBD is intestinal fibrosis (IF). It is the result of the chronic inflammation of intestinal tissue. IF causes narrowing of the intestinal lumen and potential stricture formation. For the study of the cellular and molecular mechanism of IF in IBD adequate animal models are lacking. Our aim is to develop an ex vivo model for IF by using human and rodent precision-cut intestinal slice (PCIS). In PCIS all cell types are present in their original tissue-matrix environment and can be used as a model to study the early onset of IF. Methods: Rat, mouse and human jejunum were excised and prepared as a segment embedded in agarose. PCIS (estimated 300-400 mm) was prepared and incubated up to 24 hr (rat) or 48 hr (human and mouse). ATP content of the PCIS was used to assess the general viability. Moreover, morphology (rat and human) was evaluated. The gene expression of different fibrosis markers including Pro-Collagen 1 A1 (COL1A1), Heat Shock Protein 47 (HSP47), alpha-Smooth Muscle Actin (SMA), connective tissue growth factor (CTGF), Synaptophysin (SYN) and Fibronectin (FIB) were determined. Results: Mouse PCIS were viable up to 48 hr. However, for rat and human PCIS ATP content was decreased to 25% (24hr) and 70% (48 hr), respectively. ATP content of rat and human PCIS correlated well with morphology of the PCIS. In rat PCIS, after 24 hr, HSP47 (3.2 fold) and FIB (2.1 fold) gene expressions were significantly increased. In the presence of 5 ng/mL TGF-β, COL1A1 (1.8 fold), SMA (1.5 fold) and CTGF (2.1 fold) were significantly up-regulated compared to 24 hr control. Meanwhile HSP47 gene expression was slightly decreased (0.8 fold). Similarly, in mouse PCIS, gene expression of HSP47 (3.9 fold) and FIB (4.3 fold) was significantly increased after 48 hr. When incubated with 5 ng/mL TGF-β, COl1A1 and FIB were significantly up-regulated (2.0 fold) compared to 48 hr control, HSP47 and CTGF gene expression were slightly, but significantly increased (1.2 fold). In human PCIS, after 48 hr, HSP47 (3.5-fold) and SYN (2.5 fold) were significantly up-regulated. However, incubation of human PCIS with 5 ng/ml of TGF-β, none of the investigated fibrosis genes was affected. Conclusions: In rat, mouse and human PCIS an increase in gene expression of early-onset of fibrosis makers was found. In addition, TGF-β was able to induce fibrosis markers in rat and mouse, but not in human PCIS. Rodent and human PCIS are promising ex vivo models to study the early onset of intestinal fibrosis.

Precision-cut Intestinal Slices as In Vitro Tool for Studies on Drug Metabolism

Precision-cut Intestinal Slices as In Vitro Tool for Studies on Drug Metabolism

Precision-cut Intestinal Slices as In Vitro Tool for Studies on Drug Metabolism

The role of the intestine in drug metabolism has long been underestimated as a consequence of the technical difficulty to discern the role of the intestine from that of the liver in in vivo experiments and of the lack of in vitro models that are sufficiently viable and fully representing the physiology and anatomy of the intestine. Recently the precision-cut slice model, which is widely used for liver and kidney, was also adapted for the small and large intestine. In this review the application of precision-cut intestinal slices (PCIS) for research in drug metabolism and transport is discussed. PCIS can be prepared from animal and human tissues from all regions of the intestine allowing investigation of species differences and regional gradients of activities of metabolizing enzymes. They are viable for 8-24 h of incubation and show high activity of drug metabolizing enzymes, representative for the in vivo activity. They have been successfully used to study drug-drug interactions such as induction, inhibition and regulation of drug metabolizing enzymes, transporters and nuclear factors. Moreover they appear to be a suitable model for studies on cold preservation of donor organs for transplantation, and allow exploring inter-organ interactions by co-incubation with precision-cut slices of other organs. Their application as model for drug-induced intestinal toxicity is still in its infancy but appears to be promising. PCIS, prepared from human and animal tissues, represent a powerful translational model for drug metabolism, transport and toxicity studies and as such contributes to the reduction and replacement of animal experiments.

Precision-cut Intestinal Slices as In Vitro Tool for Studies on Drug Metabolism

The role of the intestine in drug metabolism has long been underestimated as a consequence of the technical difficulty to discern the role of the intestine from that of the liver in in vivo experiments and of the lack of in vitro models that are sufficiently viable and fully representing the physiology and anatomy of the intestine. Recently the precision-cut slice model, which is widely used for liver and kidney, was also adapted for the small and large intestine. In this review the application of precision-cut intestinal slices (PCIS) for research in drug metabolism and transport is discussed. PCIS can be prepared from animal and human tissues from all regions of the intestine allowing investigation of species differences and regional gradients of activities of metabolizing enzymes. They are viable for 8-24 h of incubation and show high activity of drug metabolizing enzymes, representative for the in vivo activity. They have been successfully used to study drug-drug interactions such as induction, inhibition and regulation of drug metabolizing enzymes, transporters and nuclear factors. Moreover they appear to be a suitable model for studies on cold preservation of donor organs for transplantation, and allow exploring inter-organ interactions by co-incubation with precision-cut slices of other organs. Their application as model for drug-induced intestinal toxicity is still in its infancy but appears to be promising. PCIS, prepared from human and animal tissues, represent a powerful translational model for drug metabolism, transport and toxicity studies and as such contributes to the reduction and replacement of animal experiments.

Rat and human intestinal slices as a model for intestinal fibrosis in Inflammatory Bowel Disease

Rat and human intestinal slices as a model for intestinal fibrosis in Inflammatory Bowel Disease

Rat intestinal precision-cut slices as an in vitro model to study xenobiotic interaction with transporters

ATP-binding cassette (ABC) proteins play key role in tissue defence by transporting metabolic waste and toxic chemicals out of the cells. Consequently, intact cell systems are required to study xenobiotic interactions with ATP-dependent transporters. The aim of the present study was to set up an intestinal precision-cut slice technique to study the interactions of ABC transporters with xenobiotics. Rat intestinal slices were incubated with verapamil, indomethacin and glibenclamide, and the ability of the above-mentioned drugs to inhibit the multidrug resistance glycoprotein (MDR) and/or multidrug-resistance-associated protein (MRP) was assessed by measuring the intracellular conversion of calcein-AM to fluorescent calcein. The ABC transporters’ inhibitors caused a time-dependent florescence increase which reached the maximum value at 30 min. Verapamil and glibenclamide promoted a concentration-dependent intracellular accumulation of calcein (IC 50 8.1 × 10 −6 M, 1.9 × 10 −4 M, respectively). The effect of glibenclamide was fully reversed by washing the slices, suggesting the reversible nature of calcein accumulation. These data suggest that the precision-cut intestinal slices are a reliable, simple and fast system to evaluate xenobiotic interactions with ABC transporters in rat and, hopefully, in human intestine.

Precision-Cut Slices as a Tool for Studying Phase II Drug Metabolism in Rat Small Intestine in Vitro

ABSTRACTA wide range of endogenous and exogenous compounds including many drugs and their metabolites are inactivated by phase II drug metabolism enzymes UDP-glucuronyltransferase and glutathione S-transferase. The aim of the study was to set up an intestinal precision-cut slice technique for evaluating phase II drug metabolism in rat small intestine. The enzyme activity of UDP-glucuronyltransferase and glutathione S-transferase was measured up to 4 h of incubation and compared with those from intestinal cells obtained by mucosa scraping technique. The results from our study suggest that the activity of both phase II drug metabolism enzymes remains constant at least for a period of 4 h. Both intestinal preparations exhibit phase II metabolic activities at similar rates, but precision-cut intestinal slices have a longer life span. They remain viable up to 24 h of incubation. Precision cut slices might be reliable and quite simple system for evaluating xenobiotic phase II metabolism in rat intestine in vitro.

The role of lithocholic acid in the regulation of bile acid detoxication, synthesis, and transport proteins in rat and human intestine and liver slices

The effects of the secondary bile acid, lithocholic acid (LCA), a VDR, FXR and PXR ligand, on the regulation of bile acid metabolism (CYP3A isozymes), synthesis (CYP7A1), and transporter proteins (MRP3, MRP2, BSEP, NTCP) as well as nuclear receptors (FXR, PXR, LXRα, HNF1α, HNF4α and SHP) were studied in rat and human precision-cut intestine and liver slices at the mRNA level. Changes due to 5 to 10 μM of LCA were compared to those of other prototype ligands for VDR, FXR, PXR and GR. LCA induced rCYP3A1 and rCYP3A9 in the rat jejunum, ileum and colon, rCYP3A2 only in the ileum, rCYP3A9 expression in the liver, and CYP3A4 in the human ileum but not in liver. LCA induced the expression of rMRP2 in the colon but not in the jejunum and ileum but did not affect rMRP3 expression along the length of the rat intestine. In human ileum slices, LCA induced hMRP3 and hMRP2 expression. In rat liver slices, LCA decreased rCYP7A1, rLXRα and rHNF4α expression, induced rSHP expression, but did not affect rBSEP or rNTCP expression; whereas in the human liver, a small but significant decrease was found for hHNF1α expression. These data suggests profound species differences in the effects of LCA on bile acid transport, synthesis and detoxification. An examination of the effects of prototype VDR, PXR, GR and FXR ligands showed that these pathways are all intact in precision cut slices and that LCA exerted VDR, PXR and FXR effects. The LCA-induced altered enzymes and transporter expressions in the intestine and liver would affect the disposition of drugs.

Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies

Precision-cut tissue slices (PCTS) are viable ex vivo explants of tissue with a reproducible, well defined thickness. They represent a mini-model of the organ under study and contain all cells of the tissue in their natural environment, leaving intercellular and cell-matrix interactions intact, and are therefore highly appropriate for studying multicellular processes. PCTS are mainly used to study the metabolism and toxicity of xenobiotics, but they are suitable for many other purposes. Here we describe the protocols to prepare and incubate rat and human liver and intestinal slices. Slices are prepared from fresh liver by making a cylindrical core using a drill with a hollow bit, from which slices are cut with a specially designed tissue slicer. Intestinal tissue is embedded in cylinders of agarose before slicing. Slices remain viable for 24 h (intestine) and up to 96 h (liver) when incubated in 6- or 12-well plates under 95% O(2)/5% CO(2) atmosphere.

A microfluidic approach for in vitro assessment of interorgan interactions in drug metabolism using intestinal and liver slices

Over the past two decades, it has become increasingly clear that the intestine, in addition to the liver, plays an important role in the metabolism of xenobiotics. Previously, we developed a microfluidic-based in vitro system for the perifusion of precision-cut liver slices for metabolism studies. In the present study, the applicability of this system for the perifusion of precision-cut intestinal slices, and for the sequential perifusion of intestinal and liver slices, all from rat, was tested to mimic the in vivo first pass situation. Intestinal and liver slices, exposed to the substrates 7-ethoxycoumarin (7-EC), 7-hydroxycoumarin (7-HC) and lidocaine (Li), exhibited similar metabolic rates in the biochip and in the well plates for periods of at least 3 h. The metabolic rate remained the same when two slices were placed in adjacent microchambers and perifused sequentially. In addition, the system has been adapted to sequentially perifuse intestinal and liver tissue slices in a two-compartment co-culture perfusion system with a continuous flow of medium. It becomes possible to direct metabolites or other excreted compounds formed by an intestinal slice in the first compartment to the second compartment containing a liver slice. The intestine does not influence liver metabolism for these substrates. The interplay between these two organs was demonstrated by exposing the slices to the primary bile acid, chenodeoxycholic acid (CDCA). CDCA induced the expression of fibroblast growth factor 15 (FGF15) in the intestinal slice, which resulted in a stronger down-regulation of the enzyme, cytochrome P450 7A1 (CYP7A1), in the liver slice in the second compartment than when the liver slice was exposed to CDCA in a single-microchamber biochip. We thus demonstrate in this paper that intestinal slices, in addition to liver slices, remain functional in the biochip under flow conditions, and that the two-microchamber biochip has great potential for the study of interorgan effects. This is the first example of the incorporation of both liver and intestinal slices in a microfluidic device. Use of this microfluidic system will improve our insight into interorgan interactions and elucidate as yet unknown mechanisms involved in toxicity, gene regulation and drug-drug interactions.

Induction of Metabolism and Transport in Human Intestine: Validation of Precision-Cut Slices as a Tool to Study Induction of Drug Metabolism in Human Intestine in Vitro

Induction of drug enzyme activity in the intestine can strongly determine plasma levels of drugs. It is therefore important to predict drug-drug interactions in human intestine in vitro. We evaluated the applicability of human intestinal precision-cut slices for induction studies in vitro. Morphological examination and intracellular ATP levels indicated tissue integrity up to 24 h of incubation, whereas in proximal jejunum slices, the metabolic rate toward most substrates remained at 40 to 50% of initial values. In colon slices, the cytochrome P450 conversions were below the detection limit, but conjugation rates remained relatively constant during incubation. The inducibility of drug-metabolizing enzymes and P-glycoprotein was evaluated using prototypical inducers for five induction pathways. beta-Naphthoflavone (aryl hydrocarbon receptor ligand) induced CYP1A1 (132-fold in colon and 362-fold in proximal jejunum) and UDP glucuronosyltransferase (UGT) 1A6 mRNA (9.8-fold in colon and 3.2-fold in proximal jejunum). In proximal jejunum, rifampicin (RIF) [pregnane X receptor (PXR) ligand] induced CYP3A4 (5.2-fold), CYP2B6 (2-fold), UGT1A6 (2.2-fold), and multidrug resistance-1 (MDR1)/ABCB1 mRNA (2.7-fold), whereas 6beta-hydroxytestosterone formation (CYP3A4) increased 2-fold. In colon, RIF induced UGT1A6 32-fold and MDR1 2.2-fold. Dexamethasone (glucocorticoid receptor and PXR ligand) induced CYP3A4 mRNA (3.5-fold) and activity (5-fold) in proximal jejunum. Phenobarbital (constitutive androstane receptor activator) induced CYP3A4 (4.1-fold, only in jejunum), CYP2B6 (4.9-fold in colon and 2.3-fold in proximal jejunum), and MDR1/ABCB1 mRNA and CYP3A4 activity (2-fold only proximal jejunum). Quercetin (nuclear factor-E2-related factor 2 activator) induced UGT1A6 mRNA (6.7-fold in colon and 2.2-fold in proximal jejunum). In conclusion, this study shows that human intestinal precision-cut slices are useful to study induction of drug-metabolizing enzymes and transporters in the human intestine.

Induction of Phase I and II Drug Metabolism in Rat Small Intestine and Colon in Vitro

The aim of this study was to evaluate drug metabolism in rat small intestinal and colon precision-cut slices during 24 h of incubation and the applicability of these slices for enzyme induction studies. Various parameters were evaluated: intracellular levels of ATP (general viability marker), alkaline phosphatase activity (specific epithelial marker), villin expression (specific epithelial marker), and metabolic rates of 7-ethoxycoumarin (CYP1A), testosterone (CYP3A and CYP2B), and 7-hydroxycoumarin (glucuronide and sulfate conjugation) conversions. ATP and villin remained constant up to, respectively, 5 and 8 h in small intestine and up to 24 h in colon. The metabolic rate remained constant in small intestinal slices up to 8 h and decreased afterward to 24 to 92%, depending on the substrate studied. The inducibility of metabolism in small intestinal and colon slices was tested with several inducers at various concentrations and incubation times. The following inducers were used: 3-methylcholanthrene, beta-naphthoflavone, indirubin, and tert-butylhydroquinone (aryl hydrocarbon receptor ligands), dexamethasone (glucocorticoid receptor/pregnane X receptor ligand) and phenobarbital (constitutive androstane receptor ligand). After incubation with inducers, metabolic rates were evaluated with 7-ethoxycoumarin and testosterone (phase I) and 7-hydroxycoumarin (phase II) as substrate. All inducers elevated the metabolic rates consistent with the available published in vivo induction data. Induction of enzyme activity was already detectable after 5 h (small intestine) and after 8 h (colon) for 3-methylcholanthrene and beta-naphthoflavone and was clearly detectable for all tested inducers after 24 h (up to 20-fold compared with noninduced controls). In conclusion, small intestinal and colon precision-cut slices are useful for metabolism and enzyme induction studies.

Innovative Methods to Study Human Intestinal Drug Metabolism in Vitro: Precision-Cut Slices Compared with Ussing Chamber Preparations

Predictive in vitro methods to investigate drug metabolism in the human intestine using intact tissue are of high importance. Therefore, we studied the metabolic activity of human small intestinal and colon slices and compared it with the metabolic activity of the same human intestinal segments using the Ussing chamber technique. The metabolic activity was evaluated using substrates to both phase I and phase II reactions: testosterone, 7-hydroxycoumarin (7HC), and a mixture of cytochrome P450 (P450) substrates (midazolam, diclofenac, coumarin, and bufuralol). In slices of human proximal jejunum, the metabolic activity of several P450-mediated and conjugation reactions remained constant up to4hof incubation. In the colon slices, conjugation rates were virtually equal to those in small intestine, whereas P450-mediated conversions occurred much slower. In both organs, morphological evaluation and ATP content implied tissue integrity within this period. P450 conversions using the Ussing chamber technique showed that the metabolic rate (sum of metabolites measured in apical, basolateral, and tissue compartments) was constant up to 3 h. For 7HC conjugations, the metabolic rate remained constant up to 4 h. The distribution of the metabolites in the compartments differed between the substrates. Overall, metabolic rates were surprisingly similar in both techniques and appear similar to or even higher than in liver. In conclusion, this study shows that both human intestinal precision-cut slices and Ussing chamber preparations provide useful tools for in vitro biotransformation studies.

Three‐dimensional slice cultures from murine fetal gut for investigations of the enteric nervous system

Three-dimensional intestinal cultures offer new possibilities for the examination of growth potential, analysis of time specific gene expression, and spatial cellular arrangement of enteric nervous system in an organotypical environment. We present an easy to produce in vitro model of the enteric nervous system for analysis and manipulation of cellular differentiation processes. Slice cultures of murine fetal colon were cultured on membrane inserts for up to 2 weeks without loss of autonomous contractility. After slice preparation, cultured tissue reorganized within the first days in vitro. Afterward, the culture possessed more than 35 cell layers, including high prismatic epithelial cells, smooth muscle cells, glial cells, and neurons analyzed by immunohistochemistry. The contraction frequency of intestinal slice culture could be modulated by the neurotransmitter serotonin and the sodium channel blocker tetrodotoxin. Coculture experiments with cultured neurospheres isolated from enhanced green fluorescent protein (eGFP) transgenic mice demonstrated that differentiating eGFP-positive neurons were integrated into the intestinal tissue culture. This slice culture model of enteric nervous system proved to be useful for studying cell-cell interactions, cellular signaling, and cell differentiation processes in a three-dimensional cell arrangement.

Intestinal O2 Consumption in Necrotizing Enterocolitis: Role of Nitric Oxide

We tested the hypothesis that inducible isoform of nitric oxide synthase (iNOS)-derived nitric oxide (NO) inhibits oxygen consumption (VO2) in human intestine resected for necrotizing enterocolitis (NEC). Each NEC resection specimen was divided into two sections based on histologic appearance: healthy or diseased. Intestine removed from infants for reasons other than NEC was used as control. The tissue injury score (0-6, with 6 indicating complete necrosis) was 0.4 +/- 0.2 in control tissue, 1.2 +/- 0.4 in NEC-healthy tissue, and 4.6 +/- 0.5 in NEC-diseased tissue. Prominent iNOS staining was present in villus enterocytes in NEC-healthy tissue but not in the other tissue types. Intestinal VO2 (per direct oximetry, in nM O2/min/g) was significantly greater in control tissue than in NEC-healthy or NEC-diseased tissues. Accumulation of NO into buffer bathing intestinal slices (in nM NO/microL/g) was greater in NEC-healthy tissue than control or NEC-diseased tissues. The specific iNOS antagonist L-Nomega-(1-iminoethyl)-lysine (L-NIL) reduced buffer NO concentration 76% and increased VO2 by 90% in NEC-healthy tissue; however, L-NIL had no effect on NO or VO2 in control or NEC-diseased tissue. Addition of exogenous NO via S-nitroso-N-acetylpenicillamine depressed VO2 in NEC-healthy and control tissues but not NEC-diseased tissue. A significant correlation was present between buffer NO concentration and VO2 in NEC-healthy tissue. We conclude that iNOS-derived NO suppresses VO2 in intestine resected for NEC that demonstrates a relatively normal histology on light microscopy.