Specificity of antibodies to sea anemone toxin III and immunogenicity of the pharmacological site of anemone and scorpion toxins

Toxin III (ATX III) of the sea anemone (Anemonia sulcata) is a polypeptide containing 27 amino acid residues. It has no sequence similarity with other toxins (ATX I and II) from the same species, or with scorpion toxins, although they apparently act in a similar manner by prolonging action potentials. The specificity of ATX III antibodies was characterized using ATX III, ATX I, native and chemically modified ATX II, and scorpion alpha-toxins. The results obtained suggest that a region of ATX III, partially or totally overlapping the pharmacological site shared with ATX I and ATX II, is immunogenic. It includes a guanidino and at least two carboxylate groups. The corresponding region is not immunogenic in ATX I and ATX II. Anti-(ATX III) antibodies recognize the similar regions of ATX I and ATX II and apparently do not recognize scorpion toxins.     

Biological significance of peptides from Anemonia sulcata

Three polypeptide toxins have been isolated from the sea anemone Anemonia sulcata and characterized: ATX I (mol wt 4702), ATX II (mol wt 4935), and ATX II (mol wt 2678). In different crustacean and amphibian preparations the toxins act primarily on the fast sodium channels, which leads to delayed inactivation of fast sodium permeability and thus increases the duration of the action potential. When applied to crustacean preparations the three toxins are nearly equally effective. However, in a comparison of the biological activities of ATX I and ATX II in myelinated nerves of the frog, ATX I seems to be inactive. It is suggested that cardiotoxicity is the primary cause of death in mammals, ATX II being more toxic than ATX I. At very low concentrations ATX II induces a pronounced positive inotropic effect in different mammalian heart preparations, which is accompanied by a prolongation of the action potential. It is suggested that the positive inotropic effect of ATX II is caused by a delayed inactivation of the fast sodium current, which leads to an increase of the sodium transient and of the pump activity of Na+,K+-ATPase. In contrast to the presynaptic mode of action on crustacean and frog nerve-muscle preparations, ATX II has a direct effect on mammalian skeletal muscle fiber membranes and induces a sodium-dependent increase of twitch responses and duration of the action potential.     

Absorption and metabolism of the mycotoxins alternariol and alternariol-9-methyl ether in Caco-2 cells in vitro

Alternariol (AOH) and alternariol-9-methyl ether (AME) are major toxins produced by fungi of the genus Alternaria. In order to simulate their in vivo intestinal absorption and metabolism, AOH and AME have been studied in differentiated Caco-2 cells and in the Caco-2 Millicell® system in vitro. AOH was found to be readily conjugated to two glucuronides and one sulfate, whereas AME gave rise to one major glucuronide and one sulfate. Whereas the glucuronides of AOH and AME were sequestered about equally well into the basolateral and the apical compartment, the sulfates of both toxins were preferentially released to the apical side. Unconjugated AOH but not AME aglycone reached the basolateral chamber. The apparent permeability coefficients (P_app values) were calculated for the aglycones as well as total mycotoxin-associated compounds using an initial apical concentration of 20 µmol/l AOH or AME. Based on these P_app values, AOH must be expected to be extensively and rapidly absorbed from the intestinal lumen in vivo and reach the portal blood both as aglycone and as glucuronide and sulfate. In contrast, intestinal absorption of AME appears to be poor and sluggish, with no AME agylcone and only AME conjugates reaching the portal blood.     

Isolation and Partial Characterization of Four Host-specific Toxins of Helminthosporium maydis (Race T)

Helminthosporium maydis, race T, produces four host-specific toxins in culture. These have been designated toxins I, II, III, and IV. A method for isolation and purification of the four toxins is presented, and the criteria of purity of preparations of toxins I, II, and III are given. Toxins I and II are chemically similar and yield the same molecular ion when subjected to mass spectrometry, while toxin III appears to be a glycoside of a compound related to toxins I and II. Toxins I, II, and III can be biologically derived from ¹⁴C-mevalonic acid or ¹⁴C-acetate, permitting preparation of ¹⁴C-labeled toxins. Some chemical, spectral, and chromatographic properties of toxins I, II, and III are presented, and these data are discussed relative to the possible structure of the three compounds. In addition, four host-specific toxins have been isolated from corn infected with H. maydis (race T). These toxins are recovered in the same fractions as toxins I, II, III, and IV using the isolation procedure described here. Three of the toxins isolated from infected corn cannot be distinguished from toxins I, II, and III on the basis of infrared spectra or chromatographic mobility.     

Fatty acids affect micellar properties and modulate vitamin D uptake and basolateral efflux in Caco-2 cells

We have recently shown that vitamin D₃ (cholecalciferol) absorption is not a simple passive diffusion but involves cholesterol transporters. As free fatty acids (FAs) modulate cholesterol intestinal absorption and metabolism, we hypothesized that FAs may also interact with vitamin D absorption. Effects of FAs were evaluated at different levels of cholecalciferol intestinal absorption. First, the physicochemical properties of micelles formed with different FAs were analyzed. The micelles were then administered to human Caco-2 cells in culture to evaluate FA effects on (i) cholecalciferol uptake and basolateral efflux and (ii) the regulation of genes coding proteins involved in lipid absorption process. Micellar electric charge was correlated with both FA chain length and degree of unsaturation. Long-chain FAs at 500 μM in mixed micelles decreased cholecalciferol uptake in Caco-2 cells. This decrease was annihilated as soon as the long-chain FAs were mixed with other FAs. Oleic acid significantly improved cholecalciferol basolateral efflux compared to other FAs. These results were partly explained by a modulation of genes coding for lipid transport proteins such as Niemann-pick C1-like 1 and scavenger receptor class B type I. The data reported here show for the first time that FAs can interact with cholecalciferol intestinal absorption at different key steps of the absorption process. Cholecalciferol intestinal absorption may thus be optimized according to oil FA composition.     

Study on the intestinal permeability of lamivudine using Caco-2 cells monolayer and Single-pass intestinal perfusion

BackgroundThe aim of this work is to investigate the intestinal permeability of lamivudine and explore its absorption mechanism.MethodCaco-2 cells monolayer and single-pass intestinal perfusion (SPIP) were selected for the investigation of lamivudine under different conditions, such as different concentration, absorption time, bidirectional transportation, and transportation with efflux transporters inhibitor. The concentration of lamivudine both in Caco-2 cells monolayer samples and SPIP samples was detected by HPLC-UV. Then the permeability parameters were calculated.ResultsThe established HPLC-UV method reach the requirements for detection. There is no statistically difference between absorption parameters of lamivudine both in Caco-2 cells monolayer and SPIP (P > 0.05) under different dose groups. After transportation with efflux transporters inhibitor, the efflux rate of lamivudine in three dose groups was significantly decreased from 2.67, 2.59 and 2.59 to 1.78, 1.61, and 1.81 respectively. Lamivudine exhibits an absorption mechanism of passive diffusion.ConclusionThe absorption of lamivudine may be related to efflux transporters. In addition, lamivudine is a moderate-permeability drug in Biopharmaceutics Classification System.     

Suppressive Effects of Dietary High Fluorine on the Intestinal Development in Broilers

Fluoride (F) is a well-recognized hazardous substance. Ingested F initially acts locally on the intestines. The small intestine plays a critical role in the digestion, absorption, and defense. In this study, therefore, we investigated the effects of fluorine on the intestinal development by light microscopy, transmission electron microscopy, and histochemistry. A total of 280 one-day-old avian broilers were randomly divided into four groups and fed on a corn-soybean basal diet as control diet (fluorine, 22.6 mg/kg) or the same basal diet supplemented with 400, 800, and 1,200 mg/kg fluorine (high fluorine groups I, II, and III) in the form of sodium fluoride for 42 days. The results showed that the intestinal gross, histological, and ultrastructural changes were observed in the high fluorine groups II and III. Meanwhile, the intestinal length, weight, viscera index, villus height, crypt depth, villus height to crypt depth ratio, diameter, muscle layer thickness, and goblet cell numbers were significantly lower (p?<?0.01 or p?<?0.05), and the intestinal diameter to villus height ratio was markedly higher (p?<?0.01 or p?<?0.05) in the high fluorine groups II and III than those in control group. In conclusion, dietary fluorine in the range of 800–1,200 mg/kg obviously altered the aforementioned parameters of the intestines, implying that the intestinal development was suppressed and the intestinal functions, such as digestion, absorption, defense, or osmoregulation were impaired in broilers.     

Autotaxin and lysophosphatidic acid stimulate intestinal cell motility by redistribution of the actin modifying protein villin to the developing lamellipodia

Autotaxin (ATX) is a potent tumor cell motogen that can produce lysophosphatidic acid (LPA) from lysophosphatidylcholine. LPA is a lipid mediator that has also been shown to modulate tumor cell invasion. Autotaxin mRNA is expressed at significant levels in the intestine. Likewise, LPA2 receptor levels have been shown to be elevated in colon cancers. The molecular mechanism of ATX/LPA-induced increase in intestinal cell migration however, remains poorly understood. Villin is an intestinal and renal epithelial cell specific actin regulatory protein that modifies epithelial cell migration. In this study we demonstrate that both Caco-2 (endogenous villin) and MDCK (exogenous villin) cells, which express primarily LPA2 receptors, show enhanced cell migration in response to ATX/LPA. ATX and LPA treatment results in the rapid formation of lamellipodia and redistribution of villin to these cell surface structures, suggesting a role for villin in regulating this initial event of cell locomotion. The LPA-induced increase in cell migration required activation of c-src kinase and downstream tyrosine phosphorylation of villin by c-src kinase. LPA stimulated cell motility was determined to be insensitive to pertussis toxin, but was regulated by activation of PLC-gamma 1. Together, our results show that in epithelial cells ATX and LPA act as strong stimulators of cell migration by recruiting PLC-gamma 1 and villin, both of which participate in the initiation of protrusion.     

Caco-2 Cell Acquisition of Dietary Iron(III) Invokes a Nanoparticulate Endocytic Pathway

Dietary non-heme iron contains ferrous [Fe(II)] and ferric [Fe(III)] iron fractions and the latter should hydrolyze, forming Fe(III) oxo-hydroxide particles, on passing from the acidic stomach to less acidic duodenum. Using conditions to mimic the in vivo hydrolytic environment we confirmed the formation of nanodisperse fine ferrihydrite-like particles. Synthetic analogues of these (~ 10 nm hydrodynamic diameter) were readily adherent to the cell membrane of differentiated Caco-2 cells and internalization was visualized using transmission electron microscopy. Moreover, Caco-2 exposure to these nanoparticles led to ferritin formation (i.e., iron utilization) by the cells, which, unlike for soluble forms of iron, was reduced (p=0.02) by inhibition of clathrin-mediated endocytosis. Simulated lysosomal digestion indicated that the nanoparticles are readily dissolved under mildly acidic conditions with the lysosomal ligand, citrate. This was confirmed in cell culture as monensin inhibited Caco-2 utilization of iron from this source in a dose dependent fashion (p<0.05) whilet soluble iron was again unaffected. Our findings reveal the possibility of an endocytic pathway for acquisition of dietary Fe(III) by the small intestinal epithelium, which would complement the established DMT-1 pathway for soluble Fe(II).     

Effects of pH and temperature on cardioactive polypeptides from sea anemones: a 1H-NMR study

The effects of pH and temperature on the 300-MHz ¹H-nmr spectra of three cardioactive polypeptides from sea anemones, anthopleurin-A from Anthopleura xanthogrammica (AP-A) and Anemonia sulcata toxins I and II (ATX I and II), are described. AP-A and ATX II exhibit major spectral heterogeneity. Evidence from the pH and temperature studies and from high performance liquid chromatography indicates that this heterogeneity is conformational rather than chemical in origin. By contrast, purified isotoxins of ATX I show no evidence of conformational heterogeneity. The pKa values of most of the ionizable groups in these polypeptides are not strongly perturbed by interactions in the tertiary structure, with the exception of one of the Asp carboxylates, which has a pKa of ? 2 in AP-A and ATX II and 3.0 in ATX I. Protonation of this carboxylate, suggested to be Asp-9, leads to a conformational change in all three molecules. All three polypeptides are thermally stable, showing some conformational changes but not major unfolding at elevated temperatures.     

In vitro permeability and metabolic stability of bile pigments and the effects of hydrophilic and lipophilic modification of biliverdin

Bile pigments, including bilirubin and biliverdin are tetrapyrrolic, dicarboxylic acids capable of forming conjugates at their propionic acid groups via ester or amide bonds. They possess substantial antioxidant and anti-mutagenic activities and therefore their intestinal absorption might influence the development of cardiovascular disease and cancer. The aim of this study was to investigate whether altering the physico-chemical properties of bile pigments would improve their permeability in an in vitro assay of absorption. Native and synthetically modified bile pigments were tested for gastrointestinal permeability and metabolic stability using the Caco-2 cell line. In addition, a gross measure of their toxic effects was tested in a red blood cell co-incubation assay. The apparent permeability of unconjugated bilirubin (1), bilirubin ditaurate (2) and biliverdin (3) through Caco-2 cell monolayers was determined to be 10.4+/-1.2x10(-7), 35.2+/-3.4x10(-7) and 37.0+/-1.6x10(-7) cm/s (mean+/-SD), respectively, while biliverdin diglucosamine (4), and biliverdin dioctylamine (5) were impermeable. Unconjugated bilirubin, biliverdin, bilirubin ditaurate and biliverdin diglucosamine did not decompose when incubated in Caco-2 cell homogenates, whereas biliverdin dioctylamine decomposed over time. Only unconjugated bilirubin showed toxicity towards red blood cells (> or = 1000 microM), an effect that was abolished by the addition of 40 g/L serum albumin. The data presented here suggest that bile pigments are absorbed across the Caco-2 cell monolayer and that conjugation of biliverdin to hydrophilic or lipophilic moieties decreases their absorption and can reduce their metabolic stability. In summary, exogenous bilirubin and biliverdin supplements could be absorbed across the intestinal epithelium in vivo and potentially increase circulating concentrations of these antioxidant compounds.     

In Vitro Evaluation of the Protective Role of Lactobacillus StrainsAgainst Inorganic Arsenic Toxicity

Inorganic arsenic [iAs, As(III) + As(V)] is considered a human carcinogen. Recent studies show that it has also toxic effects on the intestinal epithelium which might partly explain its systemic toxicity. The aim of this study is to evaluate the protective role of lactic acid bacteria (LAB) against the toxic effects of iAs on the intestinal epithelium. For this purpose, the human colonic cells Caco-2 were exposed to As(III) in the presence of various LAB strains or their conditioned medium. Results showed that some strains and their conditioned media partially revert the oxidative stress, the production of pro-inflammatory cytokines, the alterations of the distribution of tight junction proteins, and the cell permeability increases caused by As(III). These results show that both soluble factors secreted or resulting from LAB metabolism and cell-cell interactions are possibly involved in the beneficial effects. Therefore, some LAB strains have potential as protective agents against iAs intestinal barrier disruption.

     

Fatty acids increase paracellular absorption of aluminium across Caco-2 cell monolayers

Passive paracellular absorption, regulated by tight junctions (TJs), is the main route for absorption of poorly absorbed hydrophilic substances. Surface active substances, such as fatty acids, may enhance absorption of these substances by affecting the integrity of TJ and increasing the permeability. It has been suggested that aluminium (Al) absorption occurs mainly by the paracellular route. Herein, we investigated if physiologically relevant exposures of fully differentiated Caco-2 cell monolayers to oleic acid and docosahexaenoic acid (DHA), which are fatty acids common in food, increase absorption of Al and the paracellular marker mannitol. In an Al toxicity test, mannitol and Al absorption through Caco-2 cell monolayers were similarly modulated by Al concentrations between 1 and 30 mM, suggesting that absorption of the two compounds occurred via the same pathways. Exposure of Caco-2 cell monolayers to non-toxic concentrations of Al (2 mM) and ¹⁴C-mannitol in fatty acid emulsions (15 and 30 mM oleic acid, 5 and 10 mM DHA) caused a decreased transepithelial electrical resistance (TEER). Concomitantly, fractional absorption of Al and mannitol, expressed as percentage of apical Al and mannitol retrieved at the basolateral side, increased with increasing dose of fatty acids. Transmission electron microscopy was applied to assess the effect of oleic acid on the morphology of TJ. It was shown that oleic acid caused a less structured morphology of TJ in Caco-2 cell monolayers. Taken together our findings indicate that fatty acids common in food increase the paracellular intestinal absorption of Al. These findings may influence future risk assessment of human Al exposure.     

Stability and absorption mechanism of typical plant miRNAs in an in vitro gastrointestinal environment: basis for their cross-kingdom nutritional effects

Plant miRNAs, a group of 19–24 nt noncoding RNAs from plant foods, were recently found to have immunomodulatory and nutritional effects on mammalian and human bodies. However, how the miRNAs survive gastrointestinal (GI) environment and how the stable miRNAs are absorbed, which serve the basis for their biological functions, were not unraveled. Here, we investigated the stabilities of six typical plant miRNAs in simulated gastric and intestinal environments, and the absorption mechanisms by Caco-2 cells. The results showed that the miRNAs can survive the environment with certain concentrations. The mixture of food ingredients enhanced the stabilities of the plant miRNAs in the gastric conditions, while 2′-O-methyl modification protects the miRNAs in intestinal juice. The stabilities of the miRNAs vary significantly in the environment and are related to their secondary structures. The stable plant miRNAs can be absorbed by Caco-2 cells via clathrin- and caveolin-mediated endocytosis. Uptake of the miRNAs was sequence dependent, facilitated by NACh and TLR9, two typical receptors on cell membrane. The results suggest that some of plant miRNAs are stable in the mimic GI environment and can be absorbed by Caco-2 cells, underlying the potential of their cross-kingdom regulation effects.     

Evaluation of the use of two human cell lines for okadaic acid and DTX-1 determination by cytotoxicity assays and damage characterization.

Two human cell lines have been used, HEp-2 and (de)differentiated Caco-2, derived from a larynx and a colon carcinoma, respectively, with the aim of evaluating and characterizing the cytotoxicity of okadaic acid (OA) and related toxins. Effects of OA and dinophysistoxin-1 (DTX-1) on cell viability (neutral red uptake) and on cell morphology/cytoskeleton structure have been observed in both cell lines, though at different time exposures and with different concentrations. The morphological alteration was detected earlier than the viability inhibition in HEp-2 cells with both toxins and in Caco-2 cells with DTX-1. HEp-2 cells have shown to be more sensitive than the intestinal cell line and thus possibly suitable for screening of contaminated samples, while Caco-2 cells could be used for further investigating the possible mechanisms involved in diarrhoeic shellfish poisoning (DSP) toxins.     

A method of quantitating Paneth cell metaplasia of the stomach by image analysis

Paneth cells are one of the histologic components of intestinal metaplasia of the stomach, as are mucin-producing goblet cells. With the aid of an image quantifier, the distribution of Paneth cells histochemically labeled with acid fuchsin was analyzed for a gastrectomy specimen containing an adenocarcinoma of the intestinal type; the topographic distribution of goblet cells histochemically labeled with Alcian blue (pH 2.5) was also analyzed. The specimen was cut into 63 blocks (0.5 X 4.0 cm) in four zones; antrum (zone I), intermediate region (zone II) and fundus (zones III and IV). Paneth cells were found only in sections containing mucin-producing goblet cells. Paneth cells were found in 12.5% of the 16 sections from the antral zone I containing Alcian blue-positive goblet cells. The rates were 44.4% for the intermediate zone II and 55.5% for the distal fundic zone III. The total area occupied by Paneth cells was significantly lower in the gastric mucosa as compared to the duodenal mucosa. The "Paneth cell index" (total Paneth cell area/total goblet cell area) was highest in the duodenum, followed by the distal fundic zone III. This method of quantitating Paneth cell metaplasia of the stomach will be used to investigate the topographic distribution of those cells in populations with low and high incidences of intestinal metaplasia.     

Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells.

Monolayers of a well differentiated human intestinal epithelial cell line, Caco-2, were used as a model to study passive drug absorption across the intestinal epithelium. Absorption rate constants (expressed as apparent permeability coefficients) were determined for 20 drugs and peptides with different structural properties. The permeability coefficients ranged from approximately 5 x 10(-8) to 5 x 10(-5) cm/s. A good correlation was obtained between data on oral absorption in humans and the results in the Caco-2 model. Drugs that are completely absorbed in humans had permeability coefficients greater than 1 x 10(-6) cm/s. Drugs that are absorbed to greater than 1% but less than 100% had permeability coefficients of 0.1-1.0 x 10(-6) cm/s while drugs and peptides that are absorbed to less than 1% had permeability coefficients of less than or equal to 1 x 10(-7) cm/s. The results indicate that Caco-2 monolayers can be used as a model for studies on intestinal drug absorption.     

Type I Interferons Function as Autocrine and Paracrine Factors to Induce Autotaxin in Response to TLR Activation

Lysophosphatidic acid (LPA) is an important phospholipid mediator in inflammation and immunity. However, the mechanism of LPA regulation during inflammatory response is largely unknown. Autotaxin (ATX) is the key enzyme to produce extracellular LPA from lysophosphatidylcholine (LPC). In this study, we found that ATX was induced in monocytic THP-1 cells by TLR4 ligand lipopolysaccharide (LPS), TLR9 ligand CpG oligonucleotide, and TLR3 ligand poly(I:C), respectively. The ATX induction by TLR ligand was abolished by the neutralizing antibody against IFN-β or the knockdown of IFNAR1, indicating that type I IFN autocrine loop is responsible for the ATX induction upon TLR activation. Both IFN-β and IFN-α were able to induce ATX expression via the JAK-STAT and PI3K-AKT pathways but with different time-dependent manners. The ATX induction by IFN-β was dramatically enhanced by IFN-γ, which had no significant effect on ATX expression alone, suggesting a synergy effect between type I and type II IFNs in ATX induction. Extracellular LPA levels were significantly increased when THP-1 cells were treated with IFN-α/β or TLR ligands. In addition, the type I IFN-mediated ATX induction was identified in human monocyte-derived dendritic cells (moDCs) stimulated with LPS or poly(I:C), and IFN-α/β could induce ATX expression in human peripheral blood mononuclear cells (PBMCs) and monocytes isolated form blood samples. These results suggest that, in response to TLR activation, ATX is induced through a type I INF autocrine-paracrine loop to enhance LPA generation.     

Extracellular metabolism-dependent uptake of lysolipids through cultured monolayer of differentiated Caco-2 cells

Glycerophospholipids are known to be hydrolyzed in the intestinal lumen into free fatty acids and lysophospholipids that are then absorbed by the intestinal epithelial cells. A monolayer of enterocyte-differentiated Caco-2 cell is often used to assess the intestinal bioavailability of nutrients. In this study, we examined how differentiated Caco-2 cells process lysoglycerolipids such as lysophosphatidylcholine (LPC). Our findings were twofold. (1) Caco-2 cells secreted both a lysophospholipase A-like enzyme and a glycerophosphocholine-phosphodiesterase enzyme into the apical, but not basolateral, lumen, suggesting that food-derived LPC is converted to a free fatty acid, sn-glycerol-3-phosphate, and choline through two sequential enzymatic reactions in humans. The release of the latter enzyme was differentiation-dependent. (2) Fatty acid-releasing activities toward exogenous fluorescent LPC, lysophosphatidic acid and monoacylglycerol were shown to be higher on the apical membranes of Caco-2 cells than on the basolateral membranes. These results suggest that human intestinal epithelial cells metabolize lysoglycerolipids by two distinct mechanisms involving secreted or apical-selective expression of metabolic enzymes.     

The human intestinal epithelial cell line Caco-2; pharmacological and pharmacokinetic applications

The gastrointestinal tract remains the most popular and acceptable route of administration for drugs. It offers the great advantage of convenience and many compounds are well absorbed and thereby provide acceptable plasma concentration-time profiles. Currently there is considerable interest from the pharmaceutical industry in development of cell culture systems that would mimic the intestinal mucosa in order to evaluate strategies for investigating and/or enhancing drug absorption. The intestinal epithelial cells of primary interest, from the standpoint of drug absorption and metabolism, are the villus cells, which are fully differentiated cells. Anin vitro cell culture system consisting of a monolayer of viable, polarized and fully differentiated villus cells, similar to that found in the small intestine, would be a valuable tool in the study of drug and nutrient transport and metabolism.The Caco-2 cell line, which exhibits a well-differentiated brush border on the apical surface and tight junctions, and expresses typical small-intestinal microvillus hydrolases and nutrient transporters, has proven to be the most popularin vitro model (a) to rapidly assess the cellular permeability of potential drug candidates, (b) to elucidate pathways of drug transport (e.g., passive versus carrier mediated), (c) to assess formulation strategies designed to enhance membrane permeability, (d) to determine the optimal physicochemical characteristics for passive diffusion of drugs, and (e) to assess potential toxic effects of drug candidates or formulation components on this biological barrier. Since differentiated Caco-2 cells express various cytochrome P450 isoforms and phase II enzymes such as UDP-glucuronosyltransferases, sulfotransferases and glutathione-S-transferases, this model could also allow the study of presystemic drug metabolism.