TC7 Clone Research Articles

In vitro and in vivo evaluation of organometallic gold(I) derivatives as anticancer agents.

Alkyne gold(I) derivatives with the water soluble phosphanes PTA (1,3,5-triaza-7-phosphaadamantane) and DAPTA (3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane) were described and their anticancer potential against the colon cancer cell line Caco-2 (PD7 and TC7 clones) was studied. Strong antiproliferative effects are found, for all the new complexes, to be even more pronounced than for the reference drug cisplatin, and similar to auranofin. The interaction of these derivatives with bovine serum albumin (BSA) was studied by fluorescence spectroscopy. The types of quenching and binding constants were determined by a fluorescence quenching method. Moderate values of the binding constants are calculated for the tested derivatives indicating that these complexes can be stored and carried easily by this protein in the body. The study of the thermodynamic parameters in the case of [Au(C[triple bond, length as m-dash]CCH2Spyridine)(PTA)] points out to the presence of van der Waals interactions or hydrogen bonding between the metallic complex and the protein. In addition, the complex [Au(C[triple bond, length as m-dash]CCH2Spyridine)(PTA)] has shown inhibition in colon cancer proliferation of HTC-116-luc2 cell lines via the apoptotic pathway and S-phase arrest of the cell cycle. Intraperitoneal injection of this derivative in athymic nude mice inoculated with HTC-116-luc2 cells prolonged their survival and displayed moderate inhibition of the tumour growth with no subsequent organ (kidney and liver) damage after treatment.

Gold(I) complexes with alkylated PTA (1,3,5-triaza-7-phosphaadamantane) phosphanes as anticancer metallodrugs

New stable thiolate gold(I) derivatives containing the alkylated phosphanes [PTA–CH2Ph]Br and [PTA–CH2COOMe]Br derived from 1,3,5-triaza-7-phosphaadamantane (PTA) have been prepared by different routes of synthesis. By the use of basic media to deprotonate the corresponding thiol in the former and by transmetallation reactions from tin (IV) complexes, in the later, thus avoiding side reactions on the phosphane. Strong antiproliferative effects are observed for most of the compounds, including the chloro- and bromo precursors with the series of phosphanes derived from PTA, in human colon cancer cell lines (Caco-2, PD7 and TC7 clones). Apoptosis-induced cell death is found for all compounds, being the thiolate derivatives with [PTA–CH2Ph]Br the most effective, as shown by an annexin-V/propidium iodide double-staining assay.

S-Propargylthiopyridine Phosphane Derivatives As Anticancer Agents: Characterization and Antitumor Activity

S-Propargylthiopyridine phosphane gold(I) derivatives with the water-soluble phosphanes PTA (1,3,5-triaza-7-phosphaadamantane), DAPTA (3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane), and TPPTS (sodium triphenylphosphane trisulfonate) are described and used as metalloligands by coordination to copper(I). New heteronuclear gold(I) and copper(I) complexes of the type [Au2Cu(C≡CCH2SC5H4N)2L2] (L = PTA, DAPTA, PPh3, TPPTS) and [AuCu(C≡CCH2SC5H4N)(L)(PPh3)2]NO3 (L = PTA, DAPTA) are reported. The X-ray crystal structure of [Au(C≡CCH2SC5H4N)(PTA)] (1), which confirms the coordination of the metallic center to the alkyne unit, displays a zigzag polymeric chain with short gold–gold contacts of 3.2680(16) Å. Strong antiproliferative effects are found for most of the new complexes in human colon cancer cell lines (Caco-2, PD7, and TC7 clones), with these effects being more pronounced than for the reference drugs cisplatin and auranofin, especially for the heterodimetallic derivatives, which are markedly more active than the corresponding mononuclear precursors. Apoptosis-induced cell death is found for all compounds, as shown by an annexin-V/propidium iodide double-staining assay.

Transport of Aflatoxin M(1) in Human Intestinal Caco-2/TC7 Cells.

Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 (AFB1). After it is formed, it is secreted in the milk of mammals. Despite the potential risk of human exposure to AFM1, data reported in literature on the metabolism, toxicity, and bioavailability of this molecule are limited and out of date. The aim of the present research was to study the absorption profile of AFM1 and possible damage to tight junctions (TJ) of the intestinal Caco-2/TC7 clone grown on microporous filter supports. These inserts allowed for the separation of the apical and basolateral compartments which correspond to the in vivo lumen and the interstitial space/vascular systems of intestinal mucosa respectively. In this study, the Caco-2/TC7 cells were treated with different AFM1 concentrations (10–10,000 ng/kg) for short (40 min) and long periods of time (48 h). The AFM1 influx/efflux transport and effects on TJ were evaluated by measuring trans-epithelial electrical resistance and observing TJ protein (Zonula occludens-1 and occludin) localization. The results showed that: (i) when introduced to the apical and basolateral compartments, AFM1 was poorly absorbed by the Caco-2/TC7 cells but its transport across the cell monolayer occurred very quickly (Papp value of 105.10 ± 7.98 cm/s × 10−6). (ii) The integrity of TJ was not permanently compromised after exposure to the mycotoxin. Viability impairment or barrier damage did not occur either. The present results contribute to the evaluation of human risk exposure to AFM1, although the AFM1 transport mechanism need to be clarified.

An Exploratory Study of Two Caco-2 Cell Models for Oral Absorption: A Report on Their Within-laboratory and Between-laboratory Variability, and Their Predictive Capacity

In 2005, the European Centre for the Validation of Alternative Methods (ECVAM) sponsored a study aimed at evaluating the reproducibility (between-laboratory and within-laboratory variability) and the predictive capacity of two in vitro cellular systems--the Caco-2/ATCC parental cell line and the Caco- 2/TC7 clone--for estimating the oral fraction absorbed (Fa) in humans. Two laboratories, both of which had experience with Caco-2 cultures, participated in the study. Ten test chemicals with documented in vivo oral absorption data were selected. Atenolol, cimetidine and propranolol were included as reference compounds for low, medium and high intestinal absorption, respectively. Transport experiments were independently carried out in the two laboratories, according to an agreed protocol. The apparent permeability coefficient (Papp) was calculated in either the apical to basolateral (absorption) or the basolateral to apical (efflux) direction. To investigate the involvement of possible active transport processes, experiments were also performed in the presence of sodium azide plus 2-deoxy-D-glucose in the donor compartment. Before performing the permeability experiments, the highest concentration that did not impair barrier integrity was identified for each test chemical in both cell models, by applying the chemicals together with a marker of the paracellular pathway. In addition, barrier integrity was assessed by measuring the trans-epithelial electrical resistance. All the permeability data obtained were independently analysed. Reproducibility was assessed for the seven substances for which sufficient data were available. Within-laboratory variability was based on coefficient of variation (CV) values. Median CV values of 10.4% and 14.7% were found for the two laboratories. Concerning between-laboratory reproducibility, comparable response levels were obtained for the three reference compounds and for paracetamol, while, for the other chemicals, the results were less reproducible--in particular, for compounds known to be actively transported. The Papp values obtained for both cell lines were comparable for identical experimental conditions. Despite the limited number of substances tested, the predictive capacity was investigated by using two mathematical models available in the literature. Good estimations of the human Fa were obtained for five well-absorbed compounds, while moderately and poorly absorbed compounds were overestimated. It is proposed that a confirmatory study addressing the main results, including power considerations, would now be useful.

Caco-2/TC7 cell line characterization for intestinal absorption: How reliable is this in vitro model for the prediction of the oral dose fraction absorbed in human?

Caco-2 cell line is one of the most used in vitro model to study intestinal absorption of compounds at screening level. Several clones have been isolated from Caco-2 cell line and characterized for their activities. Among them, TC7 clone was isolated from a late passage of the parental Caco-2 line and has shown to consist of a more homogeneous population with respect to the most representative functions of the small intestinal enterocytes, with more developed intercellular junctions. On the basis of these characteristics, it was selected within the framework of the EU A-Cute-Tox project to check its suitability to predict intestinal transport. In the present study, drugs, synthetic or natural chemicals have been characterized for their absorption profile in TC7 cells cultivated on semi-permeable filters for 21 days. The absorption experiments have been performed with the highest nontoxic concentration as determined in a preliminary set of cytotoxicity tests. The apparent permeability coefficient ( P app) has been extrapolated by calculating the passage of the test compound from the donor to the receiver compartment as a time function. The samples have been collected at different time intervals and the concentration of the test compounds analyzed by analytical methods (HPLC, GC, GC/MS). The P app obtained with the TC7 clone are comparable to those obtained with the parental cell line. However, some drawbacks related to the experimental system have been highlighted (i.e. low mass balance, adsorption to the plastics), on the basis of which some compounds were excluded from the analysis. In order to check the predictability of the model, a regression analysis has been performed by plotting P app values vs. the fraction absorbed in humans (FA, expressed as % of the administered dose). Additional elaborations have highlighted that the specific absorption pathway (passive, active and carrier-mediated) and other factors (i.e. efflux proteins and/or metabolic activity) can strongly affect the robustness of the prediction model. On the basis of the obtained results, TC7 clone has shown to be a model for passive diffusion as reliable as the parental cell line. However, we have remarked the non-suitability of the TC7 cells to predict intestinal absorption: (i) for highly lipophilic compounds; (ii) for poorly absorbed compounds; or (iii) when transporter-mediated routes and/or first pass metabolism are involved. The preliminary study of those factors likely influencing compound biokinetics, as well as the characterization of the cellular model with respect to metabolic and transporter competence, would help in the interpretation of data.

β-CRYPTOXANTHIN FROM CITRUS JUICES: BIOACCESSIBILITY AND UPTAKE BY CACO-2 CELL CULTURE MODEL

Beta-cryptoxanthin ([beta]-CX), belonging to the xanthophyll sub-class of carotenoids, is mainly found in citrus fruit in both free and ester forms. Due to the lack of knowledge on the intestinal uptake of [beta]-CX, especially the ester forms, our purpose was to study the bioaccessibility of carotenoids from citrus juice. Carotenoid uptake was evaluated by incorporating carotenoid-rich micelles (from the in vitro digestion) or synthetic micelles (made from synthetic lipids and carotenoids purified from citrus juice) to human intestinal cells (Caco-2 TC7 clone). The uptake efficiency of [beta]-CX esters was compared to free [beta]-CX. Our results suggested that esters were partially hydrolyzed during the in vitro digestion. The bioaccessibility of free [beta]-CX exceeded that of [beta]-carotene and [beta]-CX esters. These results showed a preferential uptake by Caco-2 cells of [beta]-carotene and free [beta]-CX compared to the two 'esters for all types of micelles tested. In the case of micelles from in vitro digestion, the uptake of [beta]-carotene, free [beta]-CX and [beta]-CX esters by Caco-2 cells was 14.3 ± 1.8; 3.9 ± 0.9; 0.7 ± 0.08 The present study provides the first report on assessment of [beta]-CX (free and esters) uptake by differentiated Caco-2 cells. (Resume d'auteur)

β-Cryptoxanthin fromCitrusjuices: assessment of bioaccessibility using anin vitrodigestion/Caco-2 cell culture model

Beta-Cryptoxanthin (beta-CX), a provitaminic carotenoid of potential interest for health, is found principally in Citrus fruit in both free and esterified forms. Little is known about the intestinal absorption of beta-CX especially with regard to the esterified forms. The aim of this study was to evaluate the absorption of free and esterified beta-CX using simulated digestion coupled with the Caco-2 model. Bioaccessibility was investigated by measuring the transfer of carotenoids from different citrus juices into micelles using an in vitro digestion system. Then, carotenoid uptake was evaluated by adding carotenoid-rich micelles (from the in vitro digestion) or synthetic micelles (made from synthetic lipids and carotenoids purified from citrus juice) to human intestinal cells (Caco-2 TC7 clone). Our results showed that beta-cryptoxanthin esters (beta-CXE) were partially hydrolysed during the in vitro digestion. The bioaccessibility of free beta-CX measured was significantly higher (40 (SD 1.05) %) than that of beta-carotene (30 (SD 1.9) %) and beta-CXE (16 (SD 1.5) %). In the same way, the incorporation of free beta-CX (27 (SD 1.01) %) into synthetic micelles exceeded (P<0.05) that of beta-carotene (10 (SD 0.7) %) and beta-CXE (8.8 (SD 0.4) %). In the case of micelles from in vitro digestion, the uptake of beta-carotene, free beta-CX and beta-CXE forms by Caco-2 cells was 14.3 (SD 1.8), 3.9 (SD 1.3), and 0.7 (SD 0.08) % respectively. These results showed a preferential uptake by Caco-2 cells of beta-carotene and free beta-CX compared with the two esters of beta-CX.

Effect of the main dietary antioxidants (carotenoids, γ-tocopherol, polyphenols, and vitamin C) on α-tocopherol absorption

(R,R,R)-alpha-tocopherol is a fat-soluble antioxidant vitamin generally ingested with other dietary antioxidants. The objective of this study was to assess whether the main dietary antioxidant classes, that is carotenoids, polyphenols, vitamin C and gamma-tocopherol, affect the intestinal absorption of alpha-tocopherol. METHODS, DESIGN AND SUBJECTS: We evaluated first the effect of different combinations of antioxidants on (R,R,R)-alpha-tocopherol absorption by a human intestinal cell line (Caco-2 clone TC7). Then we compared the effect of two doses of a dietary antioxidant (lutein) on the postprandial chylomicron alpha-tocopherol responses to an alpha-tocopherol-rich meal. Eight healthy men ate two similar meals in a random order at a 1 month interval. The meals contained 24 mg alpha-tocopherol in sunflower oil plus either 18 or 36 mg lutein. Blood samples were collected during the postprandial periods to compare chylomicron alpha-tocopherol responses. A mixture of polyphenols (gallic acid, caffeic acid, (+)-catechin and naringenin) and a mixture of carotenoids (lycopene, beta-carotene and lutein) significantly impaired alpha-tocopherol absorption in Caco-2 cells (P<0.001 and P<0.0001, respectively). The inhibitory effect of gamma-tocopherol was close to significance (P=0.055). In contrast, vitamin C had no significant effect (P=0.158). Naringenin was the only polyphenol that significantly impaired alpha-tocopherol absorption. Postprandial alpha-tocopherol response was weakest at the highest dose of lutein (616+/-280 nmol/l h vs 1001+/-287 nmol/l h). The observed extent of reduction (-38%, P=0.069) supported the inhibitory effect of carotenoids observed in the Caco-2 experiments. Naringenin, carotenoids and probably gamma-tocopherol can impair alpha-tocopherol absorption whereas vitamin C and phenolic acids have no effect.

Aflatoxin M 1 absorption and cytotoxicity on human intestinal in vitro model

Aflatoxin M 1 (AFM 1) is the principal hydroxylated Aflatoxin B 1 (AFB 1) metabolite and is detected in milk of mammals, after consumption of feed contaminated with AFB 1. As it is classified as probable human carcinogen (group 2B of the IARC), most countries have regulated its maximum allowed levels in milk in order to reduce AFM 1 risk (50 ng/kg the EU and 500 ng/kg in the USA). It was demonstrated that if AFB 1 must be converted into its reactive epoxide to exert its effects, and the protein binding may play an important role in its cytotoxicity. Conversely, the AFM 1 epoxidation in human liver microsomes is very limited and studies with human cell line (MCL5), expressing or not expressing cytochrome P450 enzymes, demonstrated a direct toxic potential of AFM 1 in absence of metabolic activation. For this reason, while AFM 1 is generally considered a detoxification product of AFB 1 relatively to carcinogenicity and mutagenicity property, this is not always true for cytotoxicity activity. Aim of this work is to evaluate the intestinal absorption of AFM 1 using a human in vitro model, the Caco-2 cell line. Either the parental Caco-2 cell line or its derived clone TC7, with higher metabolic competence, have been used. They were treated with different concentrations of AFM 1, that mirror the milk contamination level (0.3–32 nM corresponding to 10–10,000 ng/kg), either in undifferentiated or in differentiated phase of growth. After 48 h of treatment in serum free medium, a dose dependent absorption of AFM 1 has been detected in both cell lines, especially in differentiated cells, while, no appreciable effects on cell viability were observed, except for a general cellular suffering, revealed by LDH release, particularly evident in the undifferentiated cells. As well, no metabolites or AFM 1 conjugates have been detected. The present results may be crucial for the evaluation of human risk to AFM 1 exposure, in particular for children's population, due to their large use of milk and derivatives.

Scavenger Receptor Class B Type I (SR-BI) Is Involved in Vitamin E Transport across the Enterocyte

Although cellular uptake of vitamin E was initially described as a passive process, recent studies in the liver and brain have shown that SR-BI (scavenger receptor class B type I) is involved in this phenomenon. As SR-BI is expressed at high levels in the intestine, the present study addressed the involvement of SR-BI in vitamin E trafficking across enterocytes. Apical uptake and efflux of the main dietary forms of vitamin E were examined using Caco-2 TC-7 cell monolayers as a model of human intestinal epithelium. (R,R,R)-gamma-tocopherol bioavailability was compared between wild-type mice and mice overexpressing SR-BI in the intestine. The effect of vitamin E on enterocyte SR-BI mRNA levels was measured by real-time quantitative reverse transcription-PCR. Concentration-dependent curves for vitamin E uptake were similar for (R,R,R)-alpha-, (R,R,R)-gamma-, and dl-alpha-tocopherol. (R,R,R)-alpha-tocopherol transport was dependent on incubation temperature, with a 60% reduction in absorption at 4 degrees C compared with 37 degrees C (p < 0.05). Vitamin E flux in enterocytes was directed from the apical to the basal side, with a relative 10-fold reduction in the transfer process when measured in the opposite direction (p < 0.05). Co-incubation with cholesterol, gamma-tocopherol, or lutein significantly impaired alpha-tocopherol absorption. Anti-human SR-BI antibodies and BLT1 (a chemical inhibitor of lipid transport via SR-BI) blocked up to 80% of vitamin E uptake and up to 30% of apical vitamin E efflux (p < 0.05), and similar results were obtained for (R,R,R)-gamma-tocopherol. SR-BI mRNA levels were not significantly modified after a 24-h incubation of Caco-2 cells with vitamin E. Finally, (R,R,R)-gamma-tocopherol bioavailability was 2.7-fold higher in mice overexpressing SR-BI than in wild-type mice (p < 0.05). The present data show for the first time that vitamin E intestinal absorption is, at least in part, mediated by SR-BI.

Kinetic modelling of passive transport and active efflux of a fluoroquinolone across Caco-2 cells using a compartmental approach in NONMEM

The purpose was to develop a general mathematical model for estimating passive permeability and efflux transport parameters from in vitro cell culture experiments. The procedure is applicable for linear and non-linear transport of drug with time, <10 or >10% of drug transport, negligible or relevant back flow, and would allow the adequate correction in the case of relevant mass balance problems. A compartmental kinetic approach was used and the transport barriers were described quantitatively in terms of apical and basolateral clearances. The method can be applied when sink conditions are not achieved and it allows the evaluation of the location of the transporter and its binding site. In this work it was possible to demonstrate, from a functional point of view, the higher efflux capacity of the TC7 clone and to identify the apical membrane as the main resistance for the xenobiotic transport. This methodology can be extremely useful as a complementary tool for molecular biology approaches in order to establish meaningful hypotheses about transport mechanisms.

Molecular mechanisms of the naringin low uptake by intestinal Caco-2 cells

Naringin, the main flavanone of grapefruit, was reported to display numerous biological effects: antioxidant, hypocholesteremic, anti-atherogenic and favoring drug absorption. Naringin absorption mechanisms were studied in Caco-2 cells (TC7 clone). We investigated the possible involvement of several membrane transporters implicated in polyphenolic compounds intestinal transport (sodium-dependent glucose transporter 1, monocarboxylate transporter, multidrug-associated resistance proteins 1 and 2, and P-glycoprotein). Naringin was poorly absorbed by Caco-2 cells, according to its low value of apparent permeability coefficient (P(app) = 8.1 +/- 0.9 x 10(-8) cm/s). In the presence of verapamil, a specific inhibitor of P-glycoprotein, cellular uptake was increased by almost threefold after 5 min, and P(app) was doubled after 30 min. Our results indicated the involvement of P-glycoprotein, an ATP-driven efflux pump, capable of transporting naringin from the Caco-2 cell to the apical side. This phenomenon could explain, at least in part, the low absorption of this flavanone at the upper intestinal level.

Reciprocal inhibition of Cd and Pb sulfocomplexes for uptake in Caco‐2 cells

Cadmium-lead interactions for uptake were studied in the TC7 clone of human enterocytic-like Caco-2 cells as a function of inorganic metal speciation. We have previously shown that Cd uptake in these cells involves both the free cation Cd2+ and chlorocomplex (CdCln(2-n)) species. Here we show 1.9 times higher uptake levels for 109CdCln(2-n) compared to 210PbCln(2-n). Reciprocal inhibitions of chlorocomplexes were observed with a much higher inhibitory effect of Cd compared to Pb. Replacing Cl- by NO3- increased both the level of aquo ion 109Cd2+ and 109Cd accumulation. In contrast, higher levels of 210Pb2+ did not favor 210Pb uptake. For both metals, higher uptake data were recorded in the presence of SO4(2-), leading to sulfocomplex formation, compared with Cl-. Reciprocal inhibitions were minimal at high-cation levels but were significant and comparable in the presence of sulfo-complexes. We conclude that, in addition to Cd2+ (but not Pb2+), sulfocomplexes of both metals would preferentially be taken up compared to chlorocomplexes. NRAMP2 is not involved in Pb2+ uptake, and the NRAMP2-mediated Cd2+ uptake is insensitive to Pb. Uptake of Pb chlorocomplexes could involve specific mechanisms but of very low affinity, whereas uptake of Pb sulfocomplexes occurs with high affinity.

Increased functional expression of P-glycoprotein in Caco-2 TC7 cells exposed long-term to cadmium

The objective of this study was to investigate whether P-glycoprotein (P-gp) functional expression in intestinal cells is modified after long-term exposure to the food contaminant cadmium (Cd). The Caco-2 cell line, clone TC7, was first validated as a cellular model for long-term exposure to cadmium. Cytotoxicity tests after acute exposure of 24 h showed a significant concentration-dependent decrease in cellular viability at cadmium levels higher than 10 μM and led us to select the cadmium ranges for long-term exposure: 1, 5, and 10 μM. Intestinal cells were exposed to these cadmium concentrations for four consecutive weeks without inducing DNA condensation or fragmentation. In the second part of this work, we studied the functional expression of the drug efflux pump multidrug resistance P-glycoprotein after long-term exposure to cadmium by immunoblotting with the monoclonal antibody F4 and measurement of calcein-AM ± the P-gp inhibitor verapamil. Western blot analysis with the F4 antibody detected a single band of 170 to 180 kDa which is the size previously reported for P-gp. Calcein-AM assay showed that four weeks exposure of intestinal cells to 1, 5, and 10 μM Cd increased P-gp functional expression in proportion to the Cd concentration.

Control of differentiation-induced calbindin-D9k gene expression in Caco-2 cells by cdx-2 and HNF-1alpha.

Calbindin D9k (CaBP) is critical for intestinal calcium absorption; its in vivo expression is restricted to differentiated enterocytes of the small intestine. Our goal was to identify factors controlling the transcriptional regulation of this gene in the human intestine. Both the natural gene and a 4600-bp promoter construct were strongly regulated by differentiation (>100-fold) but not by treatment with 1,25(OH)2 vitamin D (<2-fold) in the Caco-2 clone TC7. Deletion-mutation studies revealed that conserved promoter sequences for cdx-2 (at -3158 bp) and hepatocyte nuclear factor (HNF)-1 (at -3131 and at -98 bp) combined to control CaBP expression during differentiation. Other putative response elements were not important for CaBP regulation in TC7 cells (CCAAT enhancer binding protein, pancreatic duodenal homebox-1 (pdx-1), a proximal cdx-2 element). Mutation of the distal HNF-1 site had the greatest impact on CaBP gene expression through disruption of HNF-1alpha binding; both basal and differentiation-mediated CaBP expression was reduced by 80%. In contrast, mutation of the distal cdx-2 element reduced only basal CaBP expression. Whereas a 60% reduction of CaBP mRNA in the duodenum of HNF-1alpha null mice confirmed the physiological importance of HNF-1alpha for CaBP gene regulation, additional studies showed that maximal CaBP expression requires the presence of both HNF-1alpha and cdx-2. Our data suggest that cdx-2 is a permissive factor that influences basal CaBP expression in enterocytes and that HNF-1alpha modulates CaBP gene expression during cellular differentiation.

Intestinal absorption and metabolism of carotenoids: insights from cell culture

Cell culture models are useful for studying intestinal absorption and metabolism of carotenoids. The human intestinal cell line, Caco-2, has been the most widely used model for these studies. The PF11 and TC7 clones of Caco-2 exhibit β-carotene-15,15 ′-oxygenase activity, a key enzyme in the conversion of carotenoids to vitamin A. Studies on the recent cloning of this enzyme are discussed. An in vitro cell culture system used to study intestinal absorption of carotenoids is presented. Under conditions mimicking the postprandial state, Caco-2 cells on membranes take up carotenoids and secrete them incorporated into chylomicrons. Both the cellular uptake and secretion of β-carotene are saturable, concentration-dependent processes. The selective absorption of all- trans β-carotene versus its cis isomers, the differential absorption of individual carotenoids, and the specific interactions between carotenoids during their absorption are discussed. The participation of a specific epithelial transporter in the intestinal absorption of carotenoids is proposed.

Different transport mechanisms for cadmium and mercury in Caco-2 cells: inhibition of Cd uptake by Hg without evidence for reciprocal effects

Cadmium/Hg interactions have been studied in the TC7 clone of the enterocytic-like Caco-2 cells to test the hypothesis that these metals may compete for intestinal transport. Comparison of the kinetic parameter values for 203Hg(II) and 109Cd(II) uptake in a serum-free medium revealed that Hg is accumulated much more rapidly and to higher concentrations. The very rapid uptake/binding step and the initial uptake rate of 109Cd were both significantly inhibited by an excess of unlabeled Cd or Hg (apparent K i for Hg of 9.3 ± 1.2 μM) without reciprocal effects. 109Cadmium uptake was highly sensitive to temperature and a significant fraction of accumulation (12%) was EDTA extractable. 203Hg uptake remained insensitive to temperature or the EDTA washing procedure. However, the uptake of both tracers was half-decreased when an excess of the respective unlabeled metal was added in the stop solution, suggesting an exchange mechanism for adsorption. Cell pretreatment with N-ethylmaleimide (NEM) led to a 30% decrease or a 73% increase in the 3-min specific transport of 109Cd when NEM was still present in or removed from the uptake medium, respectively. NEM had no effect on 203Hg uptake. Overall our results suggest the involvement of a saturable specific mechanism for Cd, which is highly sensitive to inhibition by Hg and NEM under some conditions, and a nonspecific passive diffusion for Hg. The Hg- or NEM-induced inhibition of Cd uptake likely involves a thiol-mediated reaction, but our results suggest that NEM pretreatment may activate other cellular mechanisms leading to a stimulatory effect.

Functional characterization of three clones of the human intestinal Caco-2 cell line for dietary lipid processing

We aimed to improve the use of the human intestinal Caco-2 cell line for studying dietary lipid and cholesterol processing by using isolated pure clones (Chantret et al. 1994). Three clones (TC7, PD7 and PF11) were grown as monolayers on semi-permeable filters and compared for cell viability, fatty acid and cholesterol apical uptake or basolateral secretion, apolipoprotein B-48 basolateral secretion and 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity. The TC7 clone showed the best viability upon apical incubation with mixed micelles and should be preferred for routine work. Short-term (3.0 h) rates of apical uptake of cholesterol were not different with the three clones, whereas the rate of apical uptake of oleic acid (18:1) was lower (P<0.05) with PF11 (250.6 nmol/mg) and the basolateral secretion of cholesterol and oleic acid was lower with the TC7 clone (0.40 and 29.1 nmol/mg respectively). The secretion of apolipoprotein B-48 basolaterally was about 2-fold lower than from PD7 clone. The basal levels of HMG-CoA reductase activity were significantly different (P<0.05; TC7>PF11 >PD7). The down-regulation of the enzyme activity was moderate (range 13.8-21.0%) and comparable in the presence of apical micellar cholesterol, but was much marked upon basolateral incubation with LDL (range 34.0-53.6%), especially for the PD7 clone. In conclusion, the Caco-2 clones characterized here proved to be particularly suitable for studying lipid nutrients processing. Because these three clones exhibit some different metabolic capabilities, they provide a new tool to study intestinal response to lipid nutrients.

Functional characterization of three clones of the human intestinal Caco-2 cell line for dietary lipid processing

We aimed to improve the use of the human intestinal Caco-2 cell line for studying dietary lipid and cholesterol processing by using isolated pure clones (). Three clones (TC7, PD7 and PF11) were grown as monolayers on semi-permeable filters and compared for cell viability, fatty acid and cholesterol apical uptake or basolateral secretion, apolipoprotein B-48 basolateral secretion and 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity. The TC7 clone showed the best viability upon apical incubation with mixed micelles and should be preferred for routine work. Short-term (3·0 h) rates of apical uptake of cholesterol were not different with the three clones, whereas the rate of apical uptake of oleic acid (18 : 1) was lower (P&lt;0·05) with PF11 (250·6 nmol/mg) and the basolateral secretion of cholesterol and oleic acid was lower with the TC7 clone (0·40 and 29·1 nmol/mg respectively). The secretion of apolipoprotein B-48 basolaterally was about 2-fold lower than from PD7 clone. The basal levels of HMG-CoA reductase activity were significantly different (P&lt;0·05; TC7&gt;PF11&gt;PD7). The down-regulation of the enzyme activity was moderate (range 13·8–21·0 %) and comparable in the presence of apical micellar cholesterol, but was much marked upon basolateral incubation with LDL (range 34·0–53·6 %), especially for the PD7 clone. In conclusion, the Caco-2 clones characterized here proved to be particularly suitable for studying lipid nutrients processing. Because these three clones exhibit some different metabolic capabilities, they provide a new tool to study intestinal response to lipid nutrients.