Uptake of phenoxyacetic acid derivatives into Caco-2 cells by the monocarboxylic acid transporters

The uptake mechanism of phenoxyacetic acid (PA) and its chlorine derivatives, 4-chlorophenoxyacetic acid (4-CPA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), was investigated using Caco-2 cells. The cells were incubated with PA, 4-CPA, 2,4-D or 2,4,5-T at pH 6.0 and 37 °C. The order of uptake and lipophilicity expressed by n-octanol partition coefficients were PA < 4-CPA < 2,4-D < 2,4,5-T. Incubation at 4 °C or at pH 7.4 significantly decreased these uptake. Furthermore, pretreatment with the protonophore, carbonylcyanide-p-(trifluoromethoxy) phenylhydrazone, or coincubation with benzoic acid, a typical substrate for the proton-linked monocarboxylic acid transporters (MCTs), significantly decreased the uptake of all compounds. The initial uptake rates of all compounds except PA were apparently saturable, suggesting the involvement of a carrier-mediated process. The order of uptake clearance of the compounds was the same as the order of their uptake and lipophilicity. Preloading of cells with benzoic acid significantly increased their uptake except for PA. These results suggest that the uptake of PA, 4-CPA, 2,4-D and 2,4,5-T from the apical membrane of Caco-2 cells is mediated via common MCTs shared, at least in part, with benzoic acid, and the increase in lipophilicity due to the chlor-substitution may increase uptake via the MCTs.     

Effects of nonionic surfactants on membrane transporters in Caco-2 cell monolayers

The objectives of this study were (1) to investigate the transporter inhibition activity of three nonionic surfactants on P-glycoprotein, the human intestinal peptide transporter, and the monocarboxylic acid transporter in Caco-2 cell monolayers, and (2) to evaluate the role of membrane fluidity and protein kinase C in surfactant-induced transporter inhibition. All three surfactants inhibited P-glycoprotein (P-gp). Over a range from 0 to 1 mM, Tween 80 and Cremophor EL increased apical-to-basolateral permeability (AP-BL) and decreased basolateral-to-apical (BL-AP) permeability of the P-gp substrate rhodamine 123. Vitamin E TPGS’s effect was equally large, but essentially only reduced the BL-AP permeability of rhodamine 123, and did so at a vitamin E TPGS concentration of only 0.025 mM. These P-gp inhibition effects would appear to be related to these excipients’ modulation of membrane fluidity, where Tween 80 and Cremophor EL fluidized cell lipid bilayers, while vitamin E TPGS rigidized lipid bilayers. However, among the three surfactants, only Tween 80 inhibited the peptide transporter, as measured by glycyl sarcosine permeability. Likewise, only Cremophor EL inhibited the monocarboxylic acid transporter, as measured by benzoic acid permeability. Nevertheless, at least one of these three surfactants inhibited each P-gp, the human intestinal peptide transporter, and the monocarboxylic acid transporter. A common functional feature of these three surfactants was their ability to modulate fluidity, although results indicate that even strong membrane fluidity modulation alone was not sufficient to reduce transporter activity. N-octyl glucoside, a nonionic surfactant that did not modulate membrane fluidity, did not affect transporter functioning. Protein kinase C inhibitors failed to affect rhodamine 123 and glycyl sarcosine permeability, suggesting protein kinase C inhibition was not the mechanism of transporter inhibition. These results suggest that surfactants can inhibit multiple transporters but that changes in membrane fluidity may not be a generalized mechanism to reduce transporter activity.     

Transepithelial transport of 4-chloro-2-methylphenoxyacetic acid (MCPA) across human intestinal Caco-2 cell monolayers

Mechanisms of transcellular transport of 4-chloro-2-methylphenoxyacetic acid (MCPA) across the small intestine were investigated using Caco-2 cells cultured on permeable membranes. The cell monolayers were incubated with MCPA, either from apical side at pH 6.0 or 7.4, or basolateral side at pH 7.4. The accumulation and apical-to-basolateral transport of MCPA were markedly stimulated by the acidic pH on the apical side (inwardly directed H(+) gradient), dependent on metabolic energy and inhibited by co-incubation with acetic acid or benzoic acid. Without the H(+) gradient, on the other hand, the basolateral-to-apical transport of MCPA (secretory transport) was higher than the apical-to-basolateral transport (absorptive transport), although the secretory transport of MCPA was markedly lower than the absorptive transport under the H(+) gradient. Co-incubation of MCPA with probenecid from the basolateral side significantly inhibited the accumulation and transport of MCPA, whereas co-incubation with p-aminohippuric acid did not. These results suggest that the absorptive transport of MCPA is mediated by H(+)-linked monocarboxylic acid transporters expressed on the apical membranes, while secretory transport is mediated by a probenecid-sensitive transporter expressed on the basolateral membranes of Caco-2 cell monolayers.     

Reproductive toxicity of MCPA (4-chloro-2-methylphenoxyacetic acid) in the rat

The reproductive effects of the administration of 4-chloro-2-methylphenoxyacetic acid (MCPA) to rats were evaluated through two generations, from prior to mating, throughout mating, to gestation and lactation. MCPA was administered in the diet at doses of 0, 50, 150, or 450 ppm to 25 male and female immature rats (F0 parents) for 10 weeks. F0 parents were then mated to produce a first litter (F1a), retained only until weaning, and were subsequently remated to produce a second litter, F1b. Groups of male and female F1b animals were then dosed as were their parents for 10 weeks postweaning, and the breeding was repeated to produce F2a and F2b animals. The study concluded with the F2b weanlings. MCPA was administered continuously throughout the study. Only minimal, non-treatment-related observations were noted, which included rhinorrhea (in both treated and control animals in the F0 generation) and malocclusion and alopecia (in both the F0 and F1b generations). There were no consistent dose-related effects on reproductive function for parental animals of either sex in either generation. Statistically significant differences were noted in body weights and body weight gains in the 450-ppm dose group for both male and female pups in F2a and F2b. There were no treatment-related macroscopic or microscopic observations noted for any animal in this study. The no-observable-effect level (NOEL) for reproductive function in rats administered MCPA continuously for two successive generations was determined to be 450 ppm (approximately 22 mg/kg/day). The NOEL for general systemic toxicity, based on body weight effects in adult animals in the F1b generation was 150 ppm. The NOEL for effects on the offspring of the F1b generation, manifested as reduced pup weights and pup weight gains was also 150 ppm (approximately 8 mg/kg/day). Based upon the results of this study, MCPA, administered for two generations to Crl:CD(SD)BR Albino rats, is considered not to be a reproductive toxicant.     

Uptake and transport of the ACE-inhibitor ceronapril (SQ 29852) by monolayers of human intestinal absorptive (Caco-2) cells in vitro

The angiotensin-converting enzyme (ACE)-inhibitor ceronapril (SQ 29852) is shown to be a substrate of the intestinal dipeptide transporter. Uptake by Caco-2 cells, grown as confluent monolayers, follows a major saturable pathway (K_m, 0.91 ± 0.11 mM; 90% at 1 mM) together with a minor passive component (k_J, 32.3 ± 6.6 ng (10⁶ cells)⁻¹ (20 min)⁻¹. Uptake was inhibited by competition with dipeptides such as l-AIa-l-Pro (K_i, 2.96 mM) and l-Phe-Gly (K_i, 3.84 mM) but not by cephalosporins such as cephalexin. In contrast, transport was non-saturable, flux increased linearly with concentration and data were consistent with a passive transepithelial transport mechanism. Transport profiles showed a biphasic dependence upon time with an initial flux of 0.83 ± 0.02 ng insert⁻¹ min⁻¹ (k₁) and a terminal value of 1.65 ± 0.08 ng insert⁻¹ min⁻¹ ((k₂) at 100 μM. It is concluded that the basolateral efflux is retarded so that the passive paracellular transport controls the overall transepithelial transport characteristics in the Caco-2 model. Carrier-mediated uptake into intestinal enterocytres, followed by rate-limiting basolateral efflux, may explain the extended t_max in vivo following oral administration.     

Teratological study of the herbicide 4-chloro-2-methylphenoxyacetic acid in rabbits

4-Chloro-2-methylphenoxyacetic acid (MCPA) is an aryloxyacetic acid derivative categorised as a plant hormone herbicide. The aim of the study was to evaluate the effect of MCPA on pregnant females and the prenatal development of rabbits. The substance tested was administered orally to pregnant New Zealand White rabbits from day 6 to day 27 of gestation at doses of 5, 10 and 25 mg kg(-1) day(-1). The animals were killed on day 28 of gestation and live fetuses were examined for gross, skeletal and visceral anomalies. Administration of MPCA did not induce any signs of maternal toxicity. There was a significant decrease of fetal and placental weight compared with controls at the highest dose of MPCA. No adverse effect of the substance tested was seen on uterine content variables, e.g. corpora lutea, pre-implantation and post-implantation loss, early, late resorptions, live and dead fetuses and sex ratio. Rabbit fetuses treated with the middle and highest doses of MPCA had a significantly elevated incidence of skull and pelvic bone delays. In conclusion, prenatal administration of MCPA did not exhibit a teratogenic effect on rabbit fetus development.     

Absorption,metabolism and excretion of 4-chloro-2-methylphenoxyacetic acid (MCPA) in rat and dog

1. The oral no overall adverse effect level (NOAEL) for chronic toxicity of 4-chloro-2-methylphenoxyacetic acid (MCPA) in rat is ~1.3?mg kg^-1 and in dog is 0.2 mgkg^-1. In an attempt to explain the difference in toxicology between these species, rats and dogs were orally dosed with (¹⁴C)-MCPA at 5 or 100?mg kg^-1 and plasma toxicokinetics, rates and routes of excretion and biotransformation were investigated. 2. Elimination of radioactivity in rat plasma was biphasic and in dog was monophasic. Rat eliminated radioactivity from plasma significantly faster than dog (approximate values based on total radioactivity: 5 mgkg^-1 rat: t_½dist 3.5 h, t_½elim 17.2-36.2 h, AUC_(0-∞) 230 µg equiv h g^-1; 5 mgkg^-1 dog: t_½47 h, AUC_(0-∞) 2500 µg equiv h g^-1; 100mg kg^-1 rat: t_½dist 10 h, t_½elim 10.27-25.4 h, AUC_(0-∞) 5400 µg equiv h g^-1; 100?mg kg^-1 dog: t_½41 h, AUC_(0-∞) 20 500µg equiv h g^-1). 3. For both species, the principal route of excretion was in urine but renal elimination was notably more rapid and more extensive in rat. 4. In both rat and dog, excretion of radioactivity was mainly as MCPA and its hydroxylated metabolite hydroxymethylphenoxyacetic acid (HMCPA). In rat, both were mainly excreted as the free acids although a small proportion was conjugated. In dog, the proportion of HMCPA was increased and the majority of both species was excreted as glycine or taurine conjugates. 5. These data, along with previously published accounts, indicate that renal elimination of MCPA in dog is substantially slower than in rat resulting in disproportionate elevation of AUC (based on total radioactivity) in dog compared with rat.     

Fluoroquinolone (ciprofloxacin) secretion by human intestinal epithelial (Caco-2) cells

1. Human intestinal epithelial Caco-2 cells were used to investigate the mechanistic basis of transepithelial secretion of the fluoroquinolone antibiotic ciprofloxacin.
2. Net secretion and cellular uptake of ciprofloxacin (at 0.1 mM) were not subject to competitive inhibition by sulphate, thiosulphate, oxalate, succinate and para-amino hippurate, probenecid (10 mM), taurocholate (100 μM) or bromosulphophthalein (100 μM). Similarly tetraethylammonium and N-′methylnicotinamide (10 mM) were without effect.
3. Net secretion of ciprofloxacin was inhibited by the organic exchange inhibitor 4,4′-diisothiocyanostilbene-2-2′-disulphonic acid (DIDS, 400 μM).
4. Net secretion of ciprofloxacin was partially inhibited by 100 μM verapamil, whilst net secretion of the P-glycoprotein substrate vinblastine was totally abolished under these conditions. Ciprofloxacin secretion was unaltered after preincubation of cells with two anti-P-glycoprotein antibodies (UIC2 and MRK16), which both significantly reduced secretory vinblastine flux (measured in the same cell batch). Ciprofloxacin (3 mM) failed to inhibit vinblastine net secretion in Caco-2 epithelia, and was not itself secreted by the P-glycoprotein expressing and vinblastine secreting dog kidney cell line, MDCK.
5. Net secretion and cellular uptake of ciprofloxacin (at 0.1 mM) were not subject to alterations of either cytosolic or medium pH, or dependent on the presence of medium Na⁺, Cl⁻ or K⁺ in the bathing media.
6. The substrate specificity of the ciprofloxacin secretory transport in Caco-2 epithelia is distinct from both the renal organic anion and cation transport. A role for P-glycoprotein in ciprofloxacin secretion may also be excluded. A novel transport mechanism, sensitive to both DIDS and verapamil mediates secretion of ciprofloxacin by human intestinal Caco-2 epithelia.

     

Absorption, distribution, metabolism and excretion of 4-chloro-2-methylphenoxyacetic acid (MCPA) in rats.

The metabolism of 14C-MCPA (4-chloro-2-methylphenoxyacetic acid) in male and female rats was compared to that of 14C- MCPA dimethylamine salt (MCPA.DMA) or 14C-MCPA ethylhexyl ester (MCPA-EHE) in adsorption, distribution, metabolism and excretion studies. Compounds were administered by the oral route. The studies demonstrated the bioequivalence of the various forms of MCPA, established the extent of metabolism and metabolite identity. Following single or multiple oral administration of 5 mg/kg 14C-MCPA quantitative recovery of radioactivity, predominantly in urine, was obtained within 168 h. Rats dosed at 100 mg/kg showed similar absorption kinetics but apparent saturation of urinary excretion led to a prolonged elimination phase. MCPA was not extensively metabolised but the oxidation product HMCPA (4-chloro-2-hydroxymethylphenoxyacetic acid) was found at low levels, together with the glycine conjugate. These metabolites were more prominent shortly after dosing, suggesting that MCPA is not retained in the liver and that these metabolites may be excreted faster than MCPA itself. MCPA.DMA and MCPA-EHE were very rapidly converted into MCPA and toxicokinetics and metabolism were indistinguishable from parent compound.     

Reabsorption of neutral amino acids mediated by amino acid transporter LAT2 and TAT1 in the basolateral membrane of proximal tubule

In order to understand the renal reabsorption mechanism of neutral amino acids via amino acid transporters, we have isolated human L-type amino acid transporter 2 (hLAT2) and human T-type amino acid transporter 1 (hTAT1) in human, then, we have examined and compared the gene structures, the functional characterizations and the localization in human kidney. Northern blot analysis showed that hLAT2 mRNA was expressed at high levels in the heart, brain, placenta, kidney, spleen, prostate, testis, ovary, lymph node and the fetal liver. The hTAT1 mRNA was detected at high levels in the heart, placenta, liver, skeletal muscle, kidney, pancreas, spleen, thymus and prostate. Immunohistochemical analysis on the human kidney revealed that the hLAT2 and hTAT1 proteins coexist in the basolateral membrane of the renal proximal tubules. The hLAT2 transports all neutral amino acids and hTAT1 transports aromatic amino acids. The basolateral location of the hLAT2 and hTAT1 proteins in the renal proximal tubule as well as the amino acid transport activity of hLAT2 and hTAT1 suggests that these transporters contribute to the renal reabsorption of neutral and aromatic amino acids in the basolateral domain of epithelial proximal tubule cells, respectively. Therefore, LAT2 and TAT1 play essential roles in the reabsorption of neutral amino acids from the epithelial cells to the blood stream in the kidney. Because LAT2 and TAT1 are essential to the efficient absorption of neutral amino acids from the kidney, their defects might be involved in the pathogenesis of disorders caused by a disruption in amino acid absorption such as blue diaper syndrome.     

Regulation of serotonin transporter activity by adenosine in intestinal epithelial cells

Serotonin plays a critical role in the regulation of intestinal physiology. The serotonin transporter (SERT) expressed in the intestinal epithelium determines 5-HT availability and activity. The serotoninergic system and SERT activity have been described as being altered in chronic intestinal pathologies such as inflammatory diseases. Adenosine has also been shown to be involved in a variety of intestinal functions and to play a central role in the regulation of inflammatory responses of injured tissue. Since the modulation of SERT by adenosine in the intestine remains unknown, the aim of the present work was to study the effect of adenosine on SERT activity and expression and to determine the molecular mechanism involved. The study has been carried out using human enterocyte-like Caco-2 cells which endogenously express SERT. The results show that adenosine diminishes SERT activity in both the apical and basal membranes by acting in the intrinsic molecule with no alteration of either SERT mRNA or protein levels. The effect of adenosine appears to be mediated by A₂ receptors and activation of the cAMP/PKA signalling pathway. Moreover, the adenosine effect did not seem to involve the activation of AMP activated protein kinase. Adenosine effects are reached at high concentrations, which suggests that adenosine modulation of SERT may be significant under conditions of inflammation and tissue injury.     

Transcellular transport of aconitine across human intestinal Caco-2 cells

Aconitine (AC) is a highly toxic compound present in plants of the genus Aconitum. The transcellular transport mechanism of AC was investigated using Caco-2 cells. The flux of AC was time- and concentration-dependent in both apical-to-basolateral and the reverse direction. The efflux of AC was more than two-fold that in the opposite direction. The influx of AC was temperature-, pH- and Na⁺-dependent. Glucose markedly decreased the absorption of AC. However, the efflux of AC was temperature- and pH-dependent, but Na⁺-independent. Cyclosporin A and verapamil, both inhibitors of P-glycoprotein (P-gp), significantly decreased the efflux of AC. In addition, MK-571, an inhibitor of multidrug resistance-associated protein 2 (MRP2), exhibited the same trend but to a lesser extent. These results indicate that both the influx and efflux of AC across Caco-2 monolayers were through an active process. A pH-dependent carrier-mediated transport system was the major absorption mechanism and a sodium-dependent glucose transporter may be involved. The active efflux of AC across Caco-2 cells was mediated mainly by ABC-transporter P-gp. It is involved in reducing the toxicity of AC to organisms and is the major reasons for the poor absorption of AC in vivo.     

Cellular Uptake Mechanism of Amino Acid Ester Prodrugs in Caco-2/hPEPT1 Cells Overexpressing a Human Peptide Transporter

Purpose. This study characterized the cellular uptake mechanism and hydrolysis of the amino acid ester prodrugs of nucleoside antiviral drugs in the transiently transfected Caco-2 cells overexpressing a human intestinal peptide transporter, hPEPTl (Caco-2/hPEPTl cells). Methods. Amino acid ester prodrugs of acyclovir and AZT were synthesized and their apical membrane permeability and hydrolysis were evaluated in Caco-2/hPEPTl cells. The cellular uptake mechanism of prodrugs was investigated through the competitive inhibition study in Caco-2/hPEPTl cells. Results. L-Valyl ester of acyclovir (L-Val-ACV) was approximately ten fold more permeable across the apical membrane than acyclovir and four times more permeable than D-valyl ester of acyclovir (D-Val-ACV). Correspondingly, L-valyl ester of AZT (L- Val-AZT) exhibited three fold higher cellular uptake than AZT. Therefore, amino acid ester prodrugs significantly increased the cellular uptake of the parent drugs and exhibited the D,L-stereoselectivity. Furthermore, prodrugs were rapidly hydrolyzed to the parent drugs by the intracellular hydrolysis, following the apical membrane transport. In the inhibition studies, cephalexin and small dipeptides strongly inhibited the cellular uptake of L-Val-ACV while L-valine had no effect, indicating that the peptide transporter is primarily responsible for the apical membrane transport of L-Val-ACV. In addition, the cellular uptake of L-Val-ACV was five times higher in Caco-2/hPEPT 1 cells than the uptake in the untransfected Caco-2 cells, implying the cellular uptake of L-Val-ACV was related to the enhancement of the peptide transport activity in Caco-2/hPEPTl cells. Conclusions. Caco-2/hPEPTl system is an efficient in vitro model for the uptake study of peptidyl derivatives. Amino acid ester prodrugs significantly improved the cellular uptake of the parent drugs via peptide transport mechanism and were rapidly converted to the active parent drugs by the intracellular hydrolysis.     

Substrate Specificity and Mechanism of the Intestinal Clonidine Uptake by Caco-2 Cells

Purpose This study was performed to characterize the substrate specificity and mechanism of the intestinal clonidine transport. Methods Uptake of [³H]clonidine into Caco-2 cells was investigated. Interaction with drugs was studied in competition assays. Results Uptake of [³H]clonidine was linear for up to 2 min, Na⁺-independent, and insensitive to changes in membrane potential, but strongly H⁺-dependent. The uptake rate of clonidine was saturable with kinetic parameters of 0.5 ± 0.1 mM (K_t) and 16.6 ± 1.8 nmol/2 min per mg of protein (V_max) at an outside pH of 7.5. Many drugs such as clonidine, guanabenz, methamphetamine, imipramine, clomipramine, nortriptyline, quinine, xylazine, ephedrine, and diphenhydramine strongly inhibited the [³H]clonidine uptake with K_i values between 0.15 and 1 mM. Conclusions Clonidine is transported by a carrier-mediated process. Substrate specificity and mechanism are very similar to the transport described in blood–brain barrier endothelial cells. The transport characteristics do not correspond to carriers for organic cations of the SLC22 family or the choline transporters CHT1 and CLT1. The system might be identical to the H⁺/tertiary amine antiporter. It interacts with a large number of both hydrophilic and lipophilic cationic drugs, and also, interestingly, with opiates.     

The effects of baicalein or baicalin on the colloidal stability of ZnO nanoparticles (NPs) and toxicity of NPs to Caco-2 cells

Recent study suggested that the presence of phytochemicals in food could interact with nanoparticles (NPs) and consequently reduce the toxicity of NPs, which has been attributed to the antioxidant properties of phytochemicals. In this study, we investigated the interactions between ZnO NPs and two flavonoids baicalein (Ba) or baicalin (Bn) as well as the influence of the interactions on the toxicity of ZnO NPs to Caco-2 cells. The antioxidant properties of Ba and Bn were confirmed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays, with Ba being stronger. However, the presence of Ba or Bn did not significantly affect cytotoxicity, intracellular superoxide or release of inflammatory cytokines of Caco-2 cells after ZnO NP exposure. When Ba was present, the cellular viability of Caco-2 cells after exposure to ZnO NPs was slightly increased, associated with a modest decrease of intracellular Zn ions, but these effects were not statistically different. Ba was more effective than Bn at changing the hydrodynamic sizes, Zeta potential and UV–Vis spectra of ZnO NPs, which indicated that Ba might increase the colloidal stability of NPs. Taken together, the results of the present study indicated that the anti-oxidative phytochemical Ba might only modestly protected Caco-2 cells from the exposure to ZnO NPs associated with an insignificant reduction of the accumulation of intracellular Zn ions. These results also indicated that when assessing the combined effects of NPs and phytochemicals to cells lining gastrointestinal tract, it might be necessary to evaluate the changes of colloidal stability of NPs altered by phytochemicals.     

Transport of the investigational anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid and its acyl glucuronide by human intestinal Caco-2 cells

5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a potent cytokine inducer, exhibited marked antitumor activity when given as multiple oral doses in mice. The aim of this study was to examine the transport of DMXAA and its acyl glucuronide (DMXAA-G) using the human Caco-2 cells. DMXAA was minimally metabolized by Caco-2 cells and both DMXAA and DMXAA-G were taken up to a minor extent by the cells. The permeability coefficient (P_app) values of DMXAA over 10–500 μM were 4 × 10⁻⁵ cm/s to 4.3 × 10⁻⁵ cm/s for both apical (AP) to basolateral (BL) and BL-AP transport, while the P_app values for the BL to AP flux of DMXAA-G were significantly greater than those for the AP to BL flux, with R_net values of 4.5–17.6 over 50–200 μM. The BL to AP active efflux of DMXAA-G followed Michaelis-Menten kinetics, with a K_m of 83.5 ± 5.5 μM, and V_max of 0.022 ± 0.001 nmol/min. The flux of DMXAA-G was energy and Na⁺-dependent and MK-571 significantly (P < 0.05) inhibited its BL to AP flux, with an estimated K_i of 130 μM. These data indicate that the transport of DMXAA across Caco-2 monolayers was through a passive process, whereas the transport of DMXAA-G was mediated by MRP1/2.     

Permeability and toxicity characteristics of L-cysteine and 2-methyl-thiazolidine-4-carboxylic acid in Caco-2 cells

Acetaldehyde is a known mutagenic substance and has been classified as a group-one carcinogen by the WHO. It is possible to bind acetaldehyde locally in the gastrointestinal (GI) tract with the semi-essential amino acid l-cysteine, which reacts covalently with acetaldehyde and forms compound 2-methyl-thiozolidine-4-carboxylic acid (MTCA). The Caco-2 cell line was used to determine the permeation of l-cysteine and MTCA, as well as the possible cell toxicity of both substances. Neither of the substances permeated through the Caco-2 cells at the concentrations used in this study, and only the highest concentration of MTCA affected the viability of the cells in the MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) test. These results showed that when l-cysteine is administered in formulations releasing it locally in the lower parts of GI tract, it is not absorbed but can react with acetaldehyde, and that neither l-cysteine nor MTCA is harmful to the cells when present locally in the upper parts of GI tract. This study also shows that MTCA is sensitive at a lower pH of 5.5. Since stable MTCA is desired in different parts of the GI tract, this observation raises concern over the influence of lower pH on l-cysteine-containing product ability to bind and eliminate carcinogenic acetaldehyde.     

Effect of thiamine on H-MPP uptake by Caco-2 cells

Recent studies on the intestinal uptake of the organic cation 1-methyl-4-phenylpyridinium (MPP⁺) showed that transport of this compound occurs through human extraneuronal monoamine transporter (hEMT). Moreover, it was recently described that alkaline phosphatase (ALP), an ecto-phosphatase anchored to the plasma membrane and able to dephosphorylate extracellular substrates or cell-surface proteins, is directly or indirectly involved in the modulation of MPP⁺ uptake by Caco-2 cells. The present study investigated a putative modulation of MPP⁺ intestinal apical uptake and ecto-ALP activity by thiamine (T⁺) and thiamine pyrophosphate (TPP, a T⁺ dietary precursor). For this purpose, we used Caco-2 cells, an enterocyte-like cell line derived from a human colonic adenocarcinoma, as an intestinal model. Ecto-ALP activity and N-[methyl-³H]-4-phenylpyridinium acetate (³H-MPP⁺) uptake were evaluated in intact Caco-2 cells. T⁺ and TPP were able to increase ecto-ALP activity, with an equal potency, and to decrease ³H-MPP⁺ apical uptake, with a similar potency. The effects of both compounds on ecto-ALP activity and ³H-MPP⁺ uptake were concentration-dependent. The results suggest that the effect of T⁺ and TPP on ecto-ALP activity may lead to inhibition of the intestinal absorption of other organic cations present in the diet. Another important conclusion is that the intestinal absorption of T⁺ may occur through hEMT, in Caco-2 cells.     

Transporters involved in apical and basolateral uptake of ceftibuten into Caco-2 cells

Ceftibuten uptake from the apical and basolateral side of Caco-2 cells grown on transwells was studied. Uptake into the cells showed concentration dependent saturation. The apical transporter(s) showed a higher capacity and lower affinity for ceftibuten than the basolateral transporter(s). Uptake was inhibited in the presence of higher pH and in the presence of 2,4-dinitro phenol (DNP). A proton gradient had a greater effect on the apical than on the basolateral transporter. Glycyl proline, a dipeptide transport system (PEPT1) substrate, inhibited ceftibuten uptake into Caco-2 cells. Benzoic acid, a monocarboxylic acid (MCT) transporter substrate also exhibited a strong inhibition of ceftibuten uptake, but acetic acid had no effect. Adipic acid inhibited apical uptake of ceftibuten but had no effect on the basolateral uptake. None of the inhibitors had a significant effect on ceftibuten uptake in absence of a pH gradient. Addition of inhibitors in presence of DNP led to a greater decrease in ceftibuten uptake, when compared to the effect of DNP alone, indicating a facilitated diffusion process. These results indicate that ceftibuten uptake in Caco-2 cells involve multiple transport pathways. Apical uptake is mediated by an energy dependent carrier-mediated process and an energy independent facilitated diffusion process. The apical transport system is different from the basolateral transporter.     

Comparison of drug transporter gene expression and functionality in Caco-2 cells from 10 different laboratories

Caco-2 cells, widely used to study carrier mediated uptake and efflux mechanisms, are known to have different properties when cultured under different conditions. In this study, Caco-2 cells from 10 different laboratories were compared in terms of mRNA expression levels of 72 drug and nutrient transporters, and 17 other target genes, including drug metabolising enzymes, using real-time PCR. The rank order of the top five expressed genes was: HPT1 > GLUT3 > GLUT5 > GST1A > OATP-B. Rank correlation showed that for most of the samples, the gene ranking was not significantly different. Functionality of transporters and the permeability of passive transport markers metoprolol (transcellular) and atenolol (paracellular) were also compared. MDR1 and PepT1 function was investigated using talinolol and Gly-Sar transport, respectively. Sulfobromophthalein (BSP) was used as a marker for MRP2 and OATP-B functionality. Atenolol permeability was more variable across laboratories than metoprolol permeability. Talinolol efflux was observed by all the laboratories, whereas only five laboratories observed significant apical uptake of Gly-Sar. Three laboratories observed significant efflux of BSP. MDR1 expression significantly correlated to the efflux ratio and net active efflux of talinolol. PepT1 mRNA levels showed significant correlation to the uptake ratio and net active uptake of Gly-Sar. MRP2 and OATP-B showed no correlation to BSP transport parameters. Heterogeneity in transporter activity may thus be due to differences in transporter expression as shown for PepT1 and MDR1 which in turn is determined by the culture conditions. Absolute expression of genes was variable indicating that small differences in culture conditions have a significant impact on gene expression, although the overall expression patterns were similar.