Cell Biology and Toxicology

, Volume 28, Issue 6, pp 369–381 | Cite as

The effect of oxidative stress upon the intestinal uptake of folic acid: in vitro studies with Caco-2 cells

  • Mafalda R. Couto
  • Pedro Gonçalves
  • Telmo Catarino
  • João R. Araújo
  • Ana Correia-Branco
  • Fátima Martel
Original Research

Abstract

Folic acid (FA) is a vitamin essential for normal cellular functions, growth, and development. Because humans cannot synthesize this micronutrient, it must be obtained from dietary sources through intestinal absorption. The intestinal tract is a major target for oxidative stress. Our aim was to investigate the effect of oxidative stress upon the uptake of FA by Caco-2 cells. Oxidative stress was induced by exposure of the cells to tert-butyl hydroperoxide (TBH) for 1 h. TBH (3,000 μM) induced an increase in biomarkers of oxidative stress, while maintaining cell viability and proliferation. In relation to the apical uptake of 3H-FA, TBH (3,000 μM) reduced the cellular accumulation of 3H-FA (10 nM), although the characteristics (kinetics, pH dependence, and inhibitory profile) of 3H-FA uptake were not changed. This effect was associated with a decrease in the mRNA steady-state levels of proton-coupled folate transporter and folate receptor alpha and of the efflux transporter multidrug resistance protein 2. Moreover, TBH (3,000 μM) did not affect the noncarrier-mediated apical uptake of 3H-FA. Finally, the effect of TBH upon 3H-FA apical uptake was not dependent on protein kinase A, protein kinase C, mitogen-activated protein kinases, phosphoinositide 3-kinase, nuclear factor kappa B, and protein tyrosine kinases, but was completely prevented by dietary polyphenols (resveratrol, quercetin, and EGCG). These results suggest that oxidative stress at the intestinal level may result in a reduction in the intestinal absorption of dietary FA and that polyphenolic dietary components may offer protection against oxidative stress-induced inhibition of intestinal FA absorption.

Keywords

Folic acid Membrane transport Oxidative stress Polyphenols tert-butyl hydroperoxide 

Introduction

Oxidative stress is caused by an imbalance between the production of free radicals and/or reactive oxygen species (ROS) and the ability of biological systems to detoxify the various forms of activated species. A major consequence of oxidative stress is damage to nucleic acid bases, lipids, and proteins, which can severely compromise cellular functions, ultimately leading to cell death. Accordingly, oxidative stress is associated with numerous human conditions, from atherosclerosis to neural degenerative diseases, inflammation, cancer, and aging (Finkel and Holbrook 2000; Klaunig and Kamendulis 2004; Stocker and Keaney 2004).

The gastrointestinal tract is a major target for oxidative stress damage due to constant exposure of ROS generated by luminal contents such as oxidized food debris, transition metals like iron and cooper, bacterial metabolites, bile acids, and salivary oxidants (Ames 1983). ROS-mediated injury to the small intestine has been demonstrated in several conditions, such as ischemia/reperfusion (Mallick et al. 2004), inflammatory bowel disease (Pravda 2005), surgical stress (Prabhu et al. 2000), radiation enteritis (Mutlu-Türkoglu et al. 2000), iron supplementation (Srigiridhar and Nair 1998), Zn deficiency (Virgili et al. 1999), and methotrexate therapy (Gao and Horie 2002).

The derivatives of the water-soluble vitamin folate (folic acid [FA]) are essential in cellular metabolism, acting as coenzymes in several single-carbon transfers involved in the biosynthesis of purines and pyrimidines, the metabolism of certain amino acids, and the initiation of protein synthesis in mitochondria (Herbert 1999). FA deficiency plays an important role in the pathogenesis of several human diseases, including macrocytic anemia, cardiovascular diseases, thromboembolytic processes, cancer, growth retardation, neural tube, and other congenital defects and various complications in pregnancy (Lucock 2000). On the other hand, FA supplementation significantly reduces the incidence of neural tube defects (Butterworth and Bendich 1996) and is associated with reductions in the risk of cardiovascular diseases (Bailey et al. 2003), Alzheimer disease (Corrada et al. 2005), and certain types of cancer (e.g., colorectal; Hubner and Houlston 2009). An adequate supply of FA is, therefore, necessary for normal human health. Because mammalian cells cannot synthesize FA, FA requirements must be entirely met by dietary sources. So, any impairment on the process of intestinal absorption of FA may create a whole-body deficiency state.

Intestinal absorption of FA occurs via a specific, concentrative, electroneutral, and acidic pH-dependent process mediated by the proton-coupled folate transporter (PCFT), a high-affinity FA transporter (Qiu et al. 2006; Zhao et al. 2009). The critical role that PCFT plays in intestinal FA absorption was established by the demonstration of loss-of-function mutations in this gene in patients with hereditary folate malabsorption (Qiu et al. 2006). However, FA is also a substrate of the reduced folate carrier 1 (RFC1), which is also present at the apical membrane of enterocytes and Caco-2 cells, and of folate receptor alpha (FRα), which is also present in the apical membrane of Caco-2 cells (Matherly and Goldman 2003; Said 2004). On the other hand, export of FA from enterocytes is dependent on transporters belonging to the ATP-binding cassette family of transporters, including the multidrug resistance-associated proteins (MRP1–MRP5) and the breast cancer resistance protein (BCRP) (Assaraf 2006).

Recent evidence shows that oxidative stress can affect the expression and/or functions of some membrane transporters (Ikemura et al. 2009). Because nothing was known concerning the effect of oxidative stress upon the intestinal absorption of FA, the aim of this work was to investigate this point, by measuring 3H-FA apical uptake by Caco-2 cells under oxidative stress conditions. Caco-2 cells are an epithelial cell line derived from human colon adenocarcinoma, which mimic the human intestinal absorptive epithelium (Delie and Rubas 1997). Oxidative stress was generated with tert-butyl hydroperoxide (TBH), a useful model compound to study mechanisms of oxidative stress injury (e.g., Aherne and O’Brien 2000; Lapshina et al. 2005; Tupe and Agte 2010). Proposed mechanisms of TBH-induced toxicity include alteration in intracellular calcium homeostasis following glutathione and protein thiol depletion, production of DNA strand breaks, onset of lipid peroxidation, and production of tert-butoxy radicals (Aherne and O’Brien 2000).

Methods

Materials

3H-folic acid ([3′,5′,7,9-3H]folic acid potassium salt; specific activity, 30 Ci/mmol) (American Radiolabeled Chemicals (ARC), St. Louis, MO, USA); acetic acid sodium salt, (E)-3-(4-methylphenylsulfonyl)-2-propenenitrile (BAY 11-7082), chelerythrine chloride, decane, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt (DIDS), dimethyl sulfoxide (DMSO), (−)-epigallocatechin-3-gallate ((−)-cis-3,3′,4′,5,5′,7-hexahydroxy-flavane-3-gallate; EGCG), 5,5-dithiobis(nitrobenzoic acid), ethanol, folic acid, fumitremorgin C (FTC; from Neosartorya fischeri), genistein (5,7-trihydroxyisoflavone), genistin (4′,5,7-trihydroxyisoflavone 7-glucoside), GSH reductase, H-89 dihydrochloride hydrate, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), indomethacin, (3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl) 1,4-dioxopyrazino [1′,2′:1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester (Ko143), 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY-294,002), 2-[N-morpholino]ethanesulfonic acid hydrate (MES), minimum essential medium, methotrexate, monensin sodium salt, 5-methyltetrahydrofolic acid disodium salt (MTHF), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADPH), penicillin/streptomycin/amphotericin B solution, probenecid, pyruvic acid sodium salt, 3,3′,4′,5,6-pentahydroxyflavone (quercetin), 3,4′,5-trihydroxy-trans-stilbene (resveratrol), serum albumin, sodium pyruvate, sulforhodamine B (SRB), TBH, thiobarbituric acid, trichloroacetic acid (TCA) sodium salt, Tris–HCl, Tris–NaOH, trypsin–EDTA solution, 2-vinylpiridine (Sigma, St. Louis, MO, USA); Alimta® (pemetrexed) (Eli Lilly and Company, Indianapolis, IN, USA); PD 98058 and SB 203580 (Research Biochemicals International, Natick, MA, USA); perchloric acid, Triton X-100 (Merck, Darmstadt, Germany).

Drugs to be tested were dissolved in H2O, decane, NaHCO3 (100 mM), DMSO, or ethanol. The final concentration of these solvents in the buffer was 1 %. Controls for these drugs were run in the presence of the respective solvent.

Caco-2 cell culture

The Caco-2 cell line was obtained from the Deutsche Sammulung von Mikroorganismen and Zellkulturen (Braunschwieg, Germany) and was used between passage numbers 20 and 65. The cells were maintained in a humidified atmosphere of 5 % CO2–95 % air and were grown in minimum essential medium containing 5.55 mM glucose and supplemented with 15 % fetal calf serum, 25 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B. Culture medium was changed every 2 to 3 days and the culture was split every 7 days. For subculturing, the cells were removed enzymatically (0.25 % trypsin–EDTA, 5 min, 37 °C), split 1:3, and subcultured in plastic culture dishes (21 cm2; Ø, 60 mm; Corning Costar, Corning, NY, USA). For quantification of cell viability, proliferation, and glutathione levels and for 3H-FA uptake studies, Caco-2 cells were seeded on 24-well plastic cell culture clusters (1.9 cm2; Ø, 15.4 mm; TPP®, Trasadingen, Switzerland) and the experiments were performed 7–10 days after the initial seeding. For the measurement of lipid peroxidation (thiobarbituric acid reactive substances [TBARS] assay), Caco-2 cells were seeded on 12-well plastic cell culture clusters (3.9 cm2; Ø, 21.4 mm; TPP®) and the experiments were performed 7–10 days after the initial seeding.

Treatment of Caco-2 cells with tert-butyl hydroperoxide

For treatment of the cells with TBH, the cell culture medium was removed and each well was washed with glucose Krebs (GK) buffer, containing in millimolars: 125 NaCl, 4.8 KCl, 1.2 MgSO4, 1.2 CaCl2, 25 NaHCO3, 1.6 KH2PO4, 0.4 K2HPO4, 5.5 glucose, and 20 HEPES, pH 7.4. Cells were then incubated for 1 h at 37 °C with increasing concentrations of TBH (1, 10, 30, 100, 1,000, and 3,000 μM) in GK buffer.

Measurement of total, oxidized, and reduced glutathione levels

Cells were treated with TBH (30, 100, 1,000 and 3,000 μM) for 1 h and then measurement of intracellular total glutathione (GSX) levels was carried out according to a previously published method (Capela et al. 2007). Briefly, cultured cells were scraped and proteins were precipitated with perchloric acid 5 %, then centrifuged for 10 min at 4 °C and the supernatant was neutralized with an equimolar solution of KHCO3. GSX contents were measured by the rate of colorimetric change of 0.7 mM 5,5-dithiobis(nitrobenzoic acid) at 415 nm in the presence of 0.4 U of GSH reductase and 0.24 mM NADPH, using a microplate reader. Oxidized glutathione (GSSG) was also quantified, using 2-vinylpiridine to block free SH groups. Reduced glutathione (GSH) levels were calculated according to the following reaction: GSX = GSH + 2 GSSG.

Measurement of lipid peroxidation (TBARS assay)

Cells were treated with TBH (30, 100, 1,000, and 3,000 μM) for 1 h and the extent of lipid peroxidation, which can be determined as the formation of malondialdehyde (MDA) after the breakdown of polyunsaturated fatty acids, was then measured by the TBARS assay. Briefly, 300 μl of cell suspension was precipitated with 200 μl of 50 % TCA and centrifuged 1 min at 6,000 rpm. Three hundred microliters of the supernatant was added with equal volume of 1 % thiobarbituric acid and the mixture was heated during 40 min at 95 °C, allowed to cool, and the absorbance measured at 535 nm (Fernandes et al. 1995).

Determination of cell viability (quantification of extracellular LDH activity)

Cells were treated with TBH (1, 10, 30, 100, 1,000, and 3,000 μM) for 1 h and then cellular leakage of the cytosolic enzyme lactate dehydrogenase (LDH) into the extracellular medium was measured spectrophotometrically by measuring the decrease in absorbance of NADH during the reduction of pyruvate to lactate, as described by Bergmeyer and Bernt (1974).

Determination of cellular proliferation (measurement of whole-cell protein (sulforhodamine B assay))

Cells were treated with TBH (1, 10, 30, 100, 1,000, and 3,000 μM) for 1 h, and afterwards, 62.5 μl of ice-cold 50 % (w/v) TCA were added to the culture medium on each well to fix cells (1 h at 4 °C in the dark). The plates were then washed five times with tap water to remove TCA. Plates were air-dried and then stained for 15 min with 0.4 % (w/v) SRB dissolved in 1 % (v/v) acetic acid. SRB was removed and cultures were rinsed four times with 1 % (v/v) acetic acid to remove residual dye. Plates were again air-dried and the bound dye was then solubilized with 375 μl of 10 mM Tris–NaOH solution (pH 10.5). The absorbance of each well was determined at 540 nm.

Measurement of 3H-FA apical uptake into Caco-2 cells

Uptake experiments were performed with Caco-2 cells incubated in GK-MES buffer (GK buffer in which HEPES is substituted by an equimolar concentration of MES), pH 5.5 (except in pH dependence experiments), after treatment with TBH or the respective solvent. First, the buffer was aspirated and the cells were washed with 0.3 ml GK-MES buffer at 37 °C; then, apical uptake of 3H-FA was initiated by the addition of 0.3 ml GK-MES buffer containing 3H-FA (10 nM, except in kinetic experiments) at 37 °C. At the end of the incubation period (6 min, except in time course experiments), incubation was stopped by placing the cells on ice and rinsing the cells with 0.3 ml ice-cold GK-MES buffer. The cells were then solubilized with 0.2 ml 0.1 % (v/v) Triton X-100 (in 5 mM Tris–HCl, pH 7.4) and placed at 37 °C overnight. Radioactivity in the cells was measured by liquid scintillation counting.

In pH dependence experiments, cells were exposed to 3H-FA (10 nM) for 6 min, at different extracellular pH values (5, 5.5, 6.5, and 7.5). For pH 5–6.5, buffer GK-MES was used. For pH 7.5, buffer GK was used.

When the effect of drugs was tested, these compounds were present during the last 20 min of treatment with TBH (or the respective solvent) and during incubation with 3H-FA.

Protein determination

The protein content of cell monolayers was determined by the method of Bradford, using serum albumin as standard.

Quantitative real-time RT-PCR

Total RNA was extracted from Caco-2 cells using the Tripure isolation reagent, according to the manufacturer’s instructions (Roche Diagnostics, Mannheim, Germany).

Before cDNA synthesis, total RNA was treated with DNase I (Ambion Inc., Austin, TX, USA) and 2 μg of the resulting DNA-free RNA was reverse transcribed using Superscript Reverse Transcriptase II and random hexamer primers (Invitrogen Corporation, Carlsbad, CA, USA) in 80 μl of final reaction volume, according to the manufacturer’s instructions. Resulting cDNA was treated with RNase H (Invitrogen Corporation, Carlsbad, CA, USA) to degrade unreacted RNA. For the quantitative real-time PCR (qRT-PCR), 2 μl of the 80-μl reverse transcription reaction mixture was used.

For the calibration curve, Caco-2 standard cDNA was diluted in five different concentrations. qRT-PCR was carried out using a LightCycler (Roche, Nutley, NJ, USA). Twenty-microliter reactions were set up in microcapillary tubes using 0.5 μM of each primer and 4 μl of SYBR Green Master Mix (LightCycler FastStart DNA MasterPlus SYBR Green I, Roche). Cycling conditions were as follows: denaturation (95 °C for 5 min), amplification and quantification [95 °C for 10 s, annealing temperature (AT) for 10 s, and 72 °C for 10 s, with a single fluorescence measurement at the end of the 72 °C for 10 s segment] repeated 55 times, a melting curve program [(AT + 10)°C for 15 s and 95 °C with a heating rate of 0.1 °C/s and continuous fluorescence measurement], and a cooling step to 40 °C for 30 s. ATs and sequence of primers are indicated in Table 1. Data were analyzed using LightCycler® 4.05 analysis software (Roche, Mannheim, Germany).
Table 1

Primers used in qRT-PCR

Gene name

Primer sequence (5′–3′)

AT (°C)

MRP2

Fwd: CCT TCA CCT CCA TTA CCC T

60

Rev: GTA CTT CTC TAG CCG CTC TGT

RFC1

Fwd: GCC CAT CGC CAC CTT TCA GAT

65

Rev: CCG CAC GTC CGA GAC AAT GA

FRα

Fwd: CAA GTT GCA TGA GCA GTG TCG

59

Rev: CAC AGT GGT TCC AGT TGA ATC

PCFT

Fwd: ATG CAG CTT TCT GCT TTG GT

59

Rev: GGA GCC ACA TAG AGC TGG AC

β-actin

Fwd: AGA GCC TCG CCT TTG CCG AT

65

Rev: CCA TCA CGC CCT GGT GCC T

MRP2 multidrug resistance protein 2, RFC1 reduced folate carrier 1, FRα folate receptor alpha, PCFT proton-coupled folate transporter

Calculation and statistics

For the analysis of the time course of 3H-FA uptake, the parameters of Eq.1 were fitted to the experimental data by a nonlinear regression analysis, using a computer-assisted method (Muzyka et al. 2005):
$$ {{{A(t) = {k_{\text{in}}}}} \left/ {{{k_{\text{out}}}\left( {1 - {e^{{ - {k_{\text{out}}} \times t}}}} \right)}} \right.} $$
(1)
where A(t) represents the accumulation of 3H-FA at time t, kin and kout are the rate constants for inward and outward transport, respectively, and t is the incubation time. Amax corresponds to the accumulation (A(t)) at steady state (t → ∞). kin is given in picomoles per milligram protein per minute and kout in per minute. In order to obtain clearance values, kin was converted to microliters per milligram protein per minute.

For the analysis of the saturation curve of 3H-FA uptake, the parameters of the Michaelis–Menten equation were fitted to the experimental data by a nonlinear regression analysis using a computer-assisted method (Muzyka et al. 2005).

Arithmetic means are given with the standard error of the mean (SEM), and geometric means are given with the 95 % confidence limits. Statistical analysis of the difference between various groups was evaluated by the analysis of variance test, followed by the Student–Newman–Keuls test. Statistical analysis of the difference between two groups was evaluated with Student’s t test. Differences were considered to be significant when P < 0.05.

Results

Effect of TBH on Caco-2 oxidative stress levels

In order to assess the effect of TBH upon Caco-2 cell oxidative stress levels, the effect of increasing concentrations of TBH on the cellular levels of GSX, GSSG, and GSH were determined. Treatment of Caco-2 cells with 3,000 μM TBH for 1 h induced a significant decrease in both GSX and GSH cellular levels, although GSSG levels were not significantly affected (Fig. 1).
Fig. 1

Effect of a 1-h exposure to increasing concentrations of TBH (30, 100, 1,000, and 3,000 μM) on Caco-2 cell glutathione levels. The effect of TBH on a total glutathione (GSX), b oxidized glutathione (GSSG), and c reduced glutathione (GSH) levels is shown. Results are expressed as nanomoles per milligram protein. Shown are the arithmetic means ± SEM (n = 12–20). Asterisk significantly different from the controls

To further evaluate the effect of TBH upon Caco-2 cell oxidative stress levels, we then measured the extent of lipid peroxidation, by using the TBARS assay. The effect of treatment with TBH upon MDA levels is shown in Fig. 2, and it can be seen that 3,000 μM TBH evoked a significant increase on the cellular concentration of MDA, indicating oxidative damage to cellular lipids.
Fig. 2

Effect of a 1-h exposure to increasing concentrations of TBH (30, 100, 1,000, and 3,000 μM) on Caco-2 lipid peroxidation levels, determined by quantification of MDA, as described in the “Methods” section. Results are shown as nanomoles MDA per milligram protein (percentage of the controls). Shown are the arithmetic means ± SEM (n = 10–21). Asterisk significantly different from the controls

Effect of TBH on Caco-2 cell viability and proliferation

The effect of TBH treatment upon Caco-2 cells viability and proliferation was then determined and is shown in Fig. 3. Exposure of Caco-2 cells for 1 h to increasing concentrations of TBH caused no increase in LDH leakage, indicating that the concentrations tested did not affect cell membrane integrity (Fig. 3a). Similarly, treatment with TBH was devoid of effect upon cell proliferation, suggesting no impairment of Caco-2 cell proliferative capacity (Fig. 3b). Thus, incubation of Caco-2 cells for 1 h with TBH up to 3,000 μM evoked no cell membrane injury and maintained a regular cell cycle.
Fig. 3

Effect of a 1-h exposure to increasing concentrations of TBH (1, 10, 30, 100, 1,000, and 3,000 μM) on Caco-2 cellular viability and proliferation. a Cellular viability was determined by quantification of extracellular LDH activity, as described in the “Methods” section. Results are shown as extracellular LDH activity (percentage of the controls) (n = 11–29). b Cellular proliferation was determined by quantification of whole cellular protein with SRB, as described in the “Methods” section. Results are shown as percentage of the controls (n = 12–27). Shown are the arithmetic means ± SEM. Asterisk significantly different from the controls

Altogether, these results indicate that treatment of Caco-2 cells for 1 h with 3,000 μM TBH induces oxidative stress, while maintaining cell viability and proliferative capacity intact. So, a concentration of 3,000 μM TBH was used in subsequent experiments aimed at determining the effect of oxidative stress upon 3H-FA uptake by Caco-2 cells.

Effect of TBH upon 3H-FA apical uptake by Caco-2 cells

Time course of uptake

In a first series of experiments, we determined the time course of 3H-FA (10 nM) accumulation in Caco-2 cells (Fig. 4). For this, cells were incubated at 37 °C with 10 nM 3H-FA for various periods of time, in the absence or presence of a previous treatment with 3,000 μM TBH for 1 h. Caco-2 cells subjected to treatment with TBH showed a reduced accumulation of 3H-FA over time, as compared with the controls. Analysis of the time course of 3H-FA uptake showed a marked difference in the Amax between control cells and cells treated with 3,000 μM TBH (5.68 ± 0.24 vs. 2.85 ± 0.12 pmol mg−1 protein, respectively; n = 6).
Fig. 4

Time course of 3H-FA uptake by Caco-2 cells. Caco-2 cells were incubated at 37 °C with 10 nM 3H-FA, at pH 5.5, in the absence (control; circles) or presence of a previous exposure to 3,000 μM TBH for 1 h (TBH; squares). Results are shown as picomoles per milligram protein. Shown are the arithmetic means ± SEM (n = 6)

Kinetics of uptake

Next, the kinetics of 3H-FA uptake by Caco-2 cells was determined, by measuring initial rates of 3H-FA uptake at increasing substrate concentrations (from 0.1 to 10 μM), both at 37 and 4 °C (corresponding to the total and noncarrier-mediated uptake of 3H-FA, respectively).

Uptake of 3H-FA at 4 °C was linear with increasing 3H-FA concentrations, and no significant differences between control and TBH-treated cells were found (results not shown). So, noncarrier-mediated uptake of 3H-FA does not appear to be affected by TBH.

Comparison of the kinetics of specific, carrier-mediated 3H-FA uptake (corresponding to uptake at 37 °C subtracted from uptake at 4 °C) in control and TBH-treated cells shows no differences both in the Vmax (65.6 ± 8.8 and 104.4 ± 38.9 pmol mg−1 protein 6 min−1, respectively) and the Km values (0.38 ± 0.31 and 2.80 ± 2.79 μM, respectively) (Fig. 5).
Fig. 5

Kinetics of specific 3H-FA uptake by Caco-2 cells. Initial rates of uptake were determined in Caco-2 cells incubated at 37 or 4 °C with increasing concentrations of 3H-FA (0.1, 1, 2.5, 5, or 10 μM) for 6 min, in the absence (control) or presence of a previous exposure to 3,000 μM TBH for 1 h (TBH). Specific uptake of 3H-FA corresponds to the difference between uptake of 3H-FA by Caco-2 cells at 37 and 4 °C. Results are shown as picomoles per milligram protein per 6 min. Shown are the arithmetic means ± SEM (n = 6–8)

pH dependence of uptake

3H-FA uptake by Caco-2 cells is known to be highly pH-dependent (Zhao et al. 2009). So, we decided to investigate the influence of TBH upon the pH dependence of 3H-FA uptake. As shown in Fig. 6, both in control and TBH-treated cells, 3H-FA uptake was strongly pH-dependent, being significantly increased at pH 5 and 5.5, when compared with pH 7.5. Uptake of 3H-FA at pH 5 and 5.5 was significantly lower in TBH-treated cells, but for pH 6.5 and 7.5, a small but significant increase in uptake of 3H-FA in TBH-treated cells was observed (Fig. 6).
Fig. 6

Effect of extracellular pH on 3H-FA uptake by Caco-2 cells. The cells were incubated at 37 °C for 6 min with 3H-FA (10 nM) at different extracellular pH values (5, 5.5, 6.5, and 7.5), in the absence (control) or presence (TBH) of a previous exposure to 3,000 μM TBH for 1 h. Results are shown as picomoles per milligram protein. Shown are the arithmetic means ± SEM (n = 6–9). Number sign significantly different from pH 7.5, asterisk significantly different from the controls

Effect of some drugs upon uptake

In order to investigate if TBH caused any changes in the pharmacological characteristics of 3H-FA uptake by Caco-2 cells, we compared the effect of some drugs known to inhibit the intestinal uptake of FA upon 3H-FA uptake in control and TBH-treated cells. For this, cells were incubated at 37 °C for 6 min with 3H-FA (10 nM) at pH 5.5, in the presence or absence of some known inhibitors of FA uptake: DIDS (100 μM), MTHF (1 μM), PTX (pemetrexed, 0.2 μM), and MTX (methotrexate, 1 μM). We verified that 3H-FA uptake was reduced by DIDS, MTHF, PTX, and MTX to the same extent in control cells and in TBH-treated cells (Fig. 7a).
Fig. 7

Effect of several compounds upon 3H-FA uptake or steady-state accumulation by Caco-2 cells. a Cells subjected to a previous exposure to 3,000 μM TBH for 1 h (TBH) or solvent (control) were incubated at 37 °C for 6 min with 3H-FA (10 nM), at pH 5.5, in the absence or presence of DIDS (100 μM), MTHF (1 μM), PTX (pemetrexed, 0.2 μM), or MTX (methotrexate, 1 μM), as described in the “Methods” section (n = 6). b Cells subjected to a previous exposure to 3,000 μM TBH (TBH) or solvent (control) for 1 h were incubated at 37 °C for 1 h with 3H-FA (10 nM), at pH 5.5, in the absence or presence of indomethacin 100 μM (INDO), probenecid 500 μM (PROB), FTC 5 μM (FTC), or Ko123 1 μM, as described in the “Methods” section (n = 8). Results are shown as the arithmetic means ± SEM. Asterisk significantly different from the controls, number sign significantly different from solvent (control) cells

FA intracellular levels depend not only on uptake mechanisms, but also on efflux mechanisms, and FA is a known substrate of some members of the ABC family of transporters. Based on the affinity of FA for different members of this family (Assaraf 2006), we decided to test the effect of two MRP inhibitors (indomethacin and probenecid) and of two BCRP inhibitors (FTC and Ko143) upon the steady-state accumulation of 3H-FA in Caco-2 cells. BCRP inhibitors were devoid of effect upon 3H-FA steady-state accumulation, both in control and TBH-treated cells (Fig. 7b). However, MRP inhibitors strongly decreased 3H-FA accumulation in control cells (to 16–20 % of the controls). Interestingly enough, the inhibitory effect of these compounds was not so marked in TBH-treated cells (Fig. 7b).

Effect of TBH upon mRNA steady-state levels of FA transporters

Next, a comparison between the mRNA expression levels of RFC1, PCFT, RFα, and MRP2 in control and TBH-treated cells was evaluated by quantitative real-time RT-PCR. Quantification of mRNA levels shows that the steady-state mRNA levels of PCFT, FRα, and MRP2 were decreased after treatment with TBH (Fig. 8).
Fig. 8

Quantification by qRT-PCR of mRNA levels of reduced folate carrier 1 (RFC1), proton-coupled folate transporter (PCFT), folate receptor alpha (FRα), and multidrug resistance protein 2 (MRP2) in Caco-2 cells after a previous exposure to 3,000 μM TBH for 1 h (TBH) or the respective solvent (control) (n = 5). Shown are the arithmetic means ± SEM corresponding to the expression of each test gene relative to β-actin. Asterisk significantly different from the controls

Effect of modulators of intracellular signaling pathways upon TBH-induced changes in 3H-FA apical uptake

We next investigated the putative involvement of signaling mechanisms in the inhibitory effect of TBH upon 3H-FA (10 nM) uptake. The signaling pathways investigated were previously reported to be activated by oxidative stress: protein kinase C (PKC), protein kinase A (PKA), members of the mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway, nuclear factor kappa B (NF-κB), and protein tyrosine kinases (PTK) (Dröge 2002; Poli et al. 2004).

No significant change in 3H-FA uptake was found with a specific PKC inhibitor (chelerythrine), and a small (10 %) decrease was observed with a specific inhibitor of PKA (H-89). The effect of TBH was not changed in the presence of any of these agents (Fig. 9).
Fig. 9

Effect of modulators of intracellular signaling pathways upon TBH-induced changes in 3H-FA uptake by Caco-2 cells. Cells subjected to a previous 1 h exposure to 3,000 μM TBH (TBH) or solvent (control) were then incubated at 37 °C for 6 min with 3H-FA (10 nM), at pH 5.5, in the absence or presence of chelerythrine 10 μM (CHEL; n = 7–11), H-89 10 μM (H89; n = 7), PD 98058 25 μM (PD; n = 8), SB 203580 25 μM (SB; n = 8), LY 294002 10 μM (LY; n = 8), BAY 11-7082 5 μM (BAY; n = 8), genistein 50 μM (GEIN; n = 10), and genistin 50 μM (GIN; n = 10–11). Shown are the arithmetic means ± SEM. Asterisk significantly different from the respective controls, number sign significantly different from TBH 3,000 μM

Next, the involvement of MAPK was studied by testing the effect of specific inhibitors of MAPK ERK1/2 (PD 98059) and of p38 MAPK (SB 203580). Both PD 98059 and SB 203580 reduced 3H-FA uptake by 20 %, but the effect of TBH was not changed in the presence of any of these agents (Fig. 9).

The specific inhibitor of the PI3K pathway (LY 294002) significantly reduced 3H-FA uptake (by 17 %), but the effect of TBH was not changed in the presence of this compound (Fig. 9). The involvement of the NF-κB signal transduction pathway was studied by testing the effect of a specific inhibitor of NF-κB signaling (BAY 11-7082). As shown in Fig. 9, BAY 11-7082 was devoid of effect upon 3H-FA uptake and also did not change the effect of TBH upon this parameter.

Finally, we also tested for the involvement of PTK by using genistein, a known PTK inhibitor. Genistein, similarly to its negative control (genistin), neither affected 3H-FA uptake nor the inhibitory effect of TBH upon 3H-FA uptake (Fig. 9).

Effect of polyphenolic compounds upon TBH-induced changes in 3H-FA apical uptake

Dietary polyphenolic compounds such as resveratrol, quercetin, and EGCG have antioxidant properties (Fraga et al. 2010) and have been shown to reduce or prevent many biological consequences of oxidative stress (Middleton et al. 2000; Fraga et al. 2010). So, we decided to investigate the ability of some polyphenols in reducing/preventing the effect of TBH upon 3H-FA uptake. Interestingly enough, none of these compounds affected 3H-FA uptake, but the effect of TBH upon 3H-FA uptake was prevented by all the polyphenols tested (namely, resveratrol, quercetin, and EGCG) (Fig. 10).
Fig. 10

Effect of polyphenolic compounds upon TBH-induced changes in 3H-FA uptake by Caco-2 cells. Cells subjected to a previous 1 h exposure to 3,000 μM TBH (TBH) or solvent (control) were then incubated at 37 °C for 6 min with 3H-FA (10 nM), at pH 5.5, in the absence or presence of resveratrol (RESV; n = 12–13), quercetin (QUER; n = 9–10), or EGCG (n = 13–16). Shown are the arithmetic means ± SEM. Asterisk significantly different from the respective controls (<0.05), number sign significantly different from TBH 3,000 μM

Discussion

The intestinal absorption of FA plays a central role in controlling and regulating FA body homeostasis. Because (1) the gastrointestinal tract is a major source of ROS and their production in excess has been linked to some chronic intestinal diseases (please see the “Introduction” section) and (2) oxidative stress is known to affect the activity and/or expression of some membrane transporters (Ikemura et al. 2009), we decided to study the effect of oxidative stress upon the intestinal absorption of FA. For this, we tested the effect of TBH upon 3H-FA uptake by a human intestinal epithelial cell line (Caco-2 cells).

The first series of experiments aimed at validating TBH as an oxidative stress-inducing compound in our cell culture system. For this, we exposed Caco-2 cells to increasing concentrations of TBH for 1 h and then quantified some biomarkers of oxidative stress. Exposure of Caco-2 cells for 1 h to 3,000 μM TBH caused a significant decrease in both GSX and GSH levels, and a significant increase in MDA levels in Caco-2 cells. A decrease in GSX and GSH levels is a good indicator of oxidative stress both in vitro and in vivo, and increased lipid peroxidation is a hallmark of oxidative stress (Dalle-Done et al. 2006), and these mechanisms have been associated with TBH-induced toxicity (Aherne and O’Brien 2000). Moreover, 3,000 μM TBH did not compromise the viability and proliferation of Caco-2 cells. Based on these results, we concluded that Caco-2 cells exposed to 3,000 μM TBH for 1 h constitute a good cellular model to study the effects of oxidative stress upon the intestinal uptake of FA, and we used TBH 3,000 μM in subsequent transport experiments.

The effect of TBH upon the apical uptake of 3H-FA by Caco-2 cells can be summarized as follows: TBH decreased the uptake of 3H-FA (10 nM), but (a) it did not affect the pH dependence of the initial rates of 3H-FA uptake, (b) it did not change inhibitor sensitivity of the initial rates of 3H-FA uptake (in relation to DIDS, MTHF, PTX, and MTX), and (c) it did not affect the kinetics of specific initial rates of 3H-FA uptake (for concentrations up to 10 μM). Moreover, TBH did not affect noncarrier-mediated uptake of 3H-FA (for concentrations up to 10 μM). The characteristics of uptake of 3H-FA in control Caco-2 cells are compatible with the involvement of PCFT, in agreement with current knowledge on the absorption of FA at the intestinal level (Qiu et al. 2006; Zhao et al. 2009). However, Caco-2 cells, differently from normal human intestinal epithelial cells, also express FRα (Matherly and Goldman 2003; Said 2004). Interestingly enough, TBH exposure caused a ±30 % decrease in the mRNA expression of both PCFT and FRα. So, the decrease in the uptake of 3H-FA by Caco-2 cells caused by TBH probably results from a decrease in the expression levels of both PCFT and FRα mRNA. Given the major role of PCFT in the intestinal absorption of FA, inhibition of PCFT by TBH will certainly affect the intestinal absorption of FA. On the other hand, inhibition of FRα will probably not interfere with the intestinal absorption of FA, as FRα does not appear to be expressed by normal human intestinal enterocytes.

Because FA intracellular levels depend not only on uptake mechanisms, but also on efflux mechanisms, and FA is a known substrate of some members of the ABC family of transporters, namely, MRP1–MRP5 and BCRP (Assaraf 2006), we decided to investigate if TBH affected the activity of MRPs and/or BCRP. BCRP inhibitors did not affect 3H-FA accumulation, both in the absence and presence of TBH, leading to the conclusion that TBH does not affect the activity of BCRP. On the other hand, MRP inhibitors (probenecid and indomethacin) strongly reduced 3H-FA accumulation (to about 20 %) in control cells and, interestingly enough, their inhibitory potency was decreased in TBH-treated cells. The reduction of 3H-FA accumulation caused by these compounds in control cells most probably results from the fact that they are able to inhibit not only efflux transporters (MRPs), but also the major FA influx transporters PCFT (Nakai et al. 2007), RFC1 (Ganapathy et al. 2004), and organic anion transporters (Miyazaki et al. 2004). Interestingly enough, TBH treatment caused a decrease in the steady-state mRNA levels not only of PCFT, but also of MRP2. So, we conclude that the reduction in the inhibitory potency of probenecid and indomethacin upon 3H-FA accumulation is the result of a decrease in the steady-state levels of PCFT and MRP2 mRNA. Contrary to our observation, some previous studies point to the conclusion of an induction of MRPs by long-term oxidative stress (Tatebe et al. 2002; Toyoda et al. 2008). Our conclusion that short-term (1 h) oxidative stress decreases MRP-mediated apical efflux of low concentrations of 3H-FA, and possibly of other MRP substrates, which includes a vast list of compounds (Zhou et al. 2008), may have serious consequences in the context of the intestinal absorption of such compounds.

Although oxidative stress is known to affect the activity and/or the expression of some membrane transporters belonging to the SLC or to the ABC families of transporters (Ikemura et al. 2009), little information is available on the detailed mechanisms involved in this effect. Oxidative stress affects several intracellular signaling cascades (Dröge 2002; Poli et al. 2004), and the activity/expression of some membrane transporters is regulated by intracellular signal transduction pathways such as PKA, PKC, and NF-κB (Ikemura et al. 2009). So, we decided to investigate the putative involvement of some intracellular signaling cascades activated by ROS (namely, PKC, PKA, MAPKs, PI3K/Akt/mTOR, NF-κB, and PTK) upon oxidative stress-induced changes in 3H-FA apical uptake. 3H-FA uptake by Caco-2 cells was found to be reduced by PKA, MAPKs (MAPK ERK1/2 and p38 MAPK), and PI3K inhibitors. Similarly to what was observed with normal rat intestinal epithelial cells (Said et al. 1997) and normal human colonic epithelial cells (Kumar et al. 1997), the PKC pathway was found to play no role in 3H-FA uptake in Caco-2 cells. However, distinctly from the normal intestinal epithelial cell lines (Kumar et al. 1997; Said et al. 1997), 3H-FA uptake by Caco-2 cells was not affected by PTK inhibition. Moreover, the inhibition of 3H-FA uptake in Caco-2 cells by PKA inhibitors agrees with the observation of Hamid and Kaur (2009), although others found no effect (Kumar et al. 1997; Said et al. 1997). In relation to TBH-induced changes in 3H-FA apical uptake, none of the intracellular signaling mechanisms investigated seem to be involved in this effect.

Several antioxidants, including dietary polyphenols, are emerging as prophylactic and therapeutic agents against disorders involving free radicals in their pathogenesis (e.g., cancer and diabetes) (Middleton et al. 2000; Fraga et al. 2010). Especially at the intestinal level, dietary phenolic compounds may help to protect the gastrointestinal tract against damage by ROS present in foods or generated within the stomach and intestine (Halliwell 2007). So, in the last part of our study, we investigated the ability of some dietary polyphenols to reduce or prevent TBH-induced changes in 3H-FA apical uptake. Interestingly enough, all the polyphenolic compounds tested (resveratrol, quercetin, and EGCG) were able to abolish the reduction in 3H-FA uptake induced by TBH.

Conclusion

The results presented in this paper show that TBH-induced oxidative stress interferes with the apical membrane transport of 3H-FA in Caco-2 cells. More specifically, TBH reduced the cellular uptake of 3H-FA (10 nM), although the characteristics (kinetics, pH dependence, and inhibitory profile) of 3H-FA uptake were not changed, and this was associated with a decrease in the mRNA steady-state levels of two influx FA transporters (PCFT and FRα) and of the efflux transporter MRP2. TBH did not affect noncarrier-mediated apical uptake of 3H-FA. The effect of TBH upon 3H-FA apical uptake was found not to be dependent on PKA, PKC, MAPKs, PI3K, NF-κB, and PTK, but was completely prevented by dietary polyphenols (resveratrol, quercetin, and EGCG). This last observation suggests that these dietary components may offer protection against oxidative stress-induced inhibition of intestinal FA absorption.

Notes

Acknowledgments

This work was supported by FCT, COMPETE, QREN, and FEDER (PTDC/SAU-OSM/102239/2008). The authors wish to thank Prof. Maria João Pinho (Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, Porto, Portugal) for her help concerning MRP2 primer design and Dr. Joana Marques (Department of Genetics, Faculty of Medicine, University of Porto, Porto, Portugal) for the donation of the β-actin primer pair.

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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Mafalda R. Couto
    • 1
  • Pedro Gonçalves
    • 1
  • Telmo Catarino
    • 1
  • João R. Araújo
    • 1
  • Ana Correia-Branco
    • 1
  • Fátima Martel
    • 1
  1. 1.Department of Biochemistry (U38-FCT), Faculty of MedicineUniversity of PortoPortoPortugal

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