In Vitro Characterization of the Intestinal Absorption of Methylmercury

Jan 7, 2014 - examines the absorption process using the Caco-2 cell line as a model of the intestinal epithelium. The results indicate that transport ...
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In Vitro Characterization of the Intestinal Absorption of Methylmercury using a Caco‑2 Cell Model Marta Vázquez, Dinoraz Vélez, and Vicenta Devesa* Instituto de Agroquímica y Tecnología de Alimentos (IATA−CSIC), Avenida Agustín Escardino 7, 46980 Paterna, Valencia, Spain

ABSTRACT: Methylmercury (CH3Hg) is one of the forms of mercury found in food, particularly in seafood. Exposure to CH3Hg is associated with neurotoxic effects during development. In addition, methylmercury has been classified by the International Agency for Research on Cancer as a possible human carcinogen. Although the diet is known to be the main source of exposure, few studies have characterized the mechanisms involved in the absorption of this contaminant. The present study examines the absorption process using the Caco-2 cell line as a model of the intestinal epithelium. The results indicate that transport across the intestinal cell monolayer in an absorptive direction occurs mainly through passive transcellular diffusion. This mechanism coexists with carrier-mediated transcellular transport, which has an active component. The participation of H+- and Na+-dependent transport was observed. Inhibition tests point to the possible participation of amino acid transporters (B0,+ system, L system, and/or y+L system) and organic anion transporters (OATs). Our study suggests the participation in CH3Hg absorption of transporters that have already been identified as being responsible for the transport of this species in other systems, although further studies are needed to confirm their participation in intestinal absorption. It should be noted that CH3Hg experiences important cellular acumulation (48−78%). Considering the toxic nature of this contaminant, this fact could affect intestinal epithelium function.

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INTRODUCTION Methylmercury (CH3Hg) is considered by the International Agency for Research on Cancer as a probable human carcinogen.1 In addition, CH3Hg is neurotoxic, particularly in the developmental stages of life, and also affects the cardiovascular, gastrointestinal, hepatobiliary, and renal systems.2 The main form of human exposure to CH3Hg is through the diet, particularly large predator fish species.3,4 Considering the health risk posed by this mercury species, the World Health Organization (WHO) has established a provisional tolerable weekly intake of 1.6 μg/kg body weight/week.5 Following ingestion, CH3Hg is quickly absorbed in the gastrointestinal tract,6 and this absorption is proportionally greater than in the case of the inorganic forms of this element. Previous studies have shown that about 95% of CH3Hg in fish ingested by humans and about 95% of methylmercuric nitrate given orally to volunteers are absorbed.7 Although the absorption rate of this mercury species is known, many aspects related to the absorption process remain unclear, including the factors that affect its absorption and the mechanisms of transport involved. © 2014 American Chemical Society

Regarding the mechanisms of transport of CH3Hg, many studies have been performed in other organs, cell types, and model systems, particularly for CH3Hg bound to thiols, such as glutathione (GSH), or to different forms of cysteine (cysteine (Cys), homocysteine (Hcys), and N-acetylcysteine (NAC)). In this context, Zalups and Ahmad8 have demonstrated the capacity of organic anion transporter 1 (OAT1) to incorporate CH3Hg−NAC to transfected renal cells. Na+-dependent neutral amino acid transporters (B0,+ system) have also been shown to participate in the transport of CH3Hg−Cys and CH3Hg−Hcys in oocytes of Xenopus laevis.9 Others have suggested the participation of Na+-independent neutral amino acid transporters 1 and 2 (LAT1 and LAT2) in the transport of CH3Hg− Cys in neural cells.10−13 Likewise, the participation of other transport systems in rat erythrocytes has been postulated, including a facilitated-diffusion D-glucose transport system.14 The extensive research made in relation to CH3Hg transport in target organs stands in contrast with the limited information Received: October 8, 2013 Published: January 7, 2014 254

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and 37 °C. Two transport directions were tested: apical−basolateral (A−B) and basolateral−apical (B−A). The standard solutions of CH3Hg were prepared from commercial CH3HgCl (1000 mg/L, Alfa Aesar, Spain) in Hanks’ balanced salt solution (HBSS) with NaHCO3 (PAA) supplemented with 10 mM HEPES (HBSS-HEPES). For analyzing the transport in the A−B direction, the cells were exposed to various concentrations of CH3Hg: 0.1, 0.5, 1, and 5 mg/L, equivalent to 0.46, 2.3, 4.6, and 23 μM, respectively. For the B−A direction, the concentrations used were 0.5 and 1 mg/L. Before starting the experiment, the cells were conditioned with HBSS-HEPES for 15 min followed by addition of the CH3Hg standard solutions to the donor compartment (apical or basal, depending on whether the test was in the A−B or B−A direction, respectively) and HEPES-HBSS to the acceptor compartment. At pre-established time points (5, 15, 30, 60, 90, and 120 min), aliquots were removed from the acceptor compartment (600 μL) and were replaced by the same volume of HBSS-HEPES. The aliquots removed at each time point were processed for mercury-content analysis as previously described.21 Likewise, mercury in the cell monolayer and in the donor medium collected at the end of the experiment was quantified. The apparent permeability coefficients (Papp; cm/s) were calculated from eq 1

available on the mechanisms involved in human intestinal absorption.2 Such studies are of great interest considering that the intestinal mucosa is the first barrier against the systemic distribution of this mercury species and therefore may influence its toxic effects. The only study in this respect, involving CH3HgCl and CH3Hg−Cys, concluded that LATs participate in the transport of CH3Hg−Cys in intestinal cells but not in the transport of CH3HgCl.15 These transporters are located in the basolateral membrane of the enterocytes, with no information being available with respect to the apical membrane. However, the basolateral membrane contains members of other families of transporters that could participate in the transport of CH3Hg, such as OAT or LATs, and that have been seen to intervene in the transport of this species in other cell types.8,10−13 The aim of the present study is to characterize the intestinal transport of CH3Hg using the Caco-2 cell line as a model of the intestinal epithelium. These cells differentiate spontaneously upon reaching confluence, giving rise to a polarized cell monolayer similar to that created by mature enterocytes. Formed monolayers have intercellular tight junctions, domas, and microvilli on the apical side and also have been found to express transporters present in the brush border membrane of absorptive cells of the small intestine.16 For this reason, they are a good model to evaluate the absorption that occurs in the small intestine.16−19 Moreover, during their differentiation, Caco-2 cells express progressively different hydrolase activities, such as sucrose isomaltase, lactase, aminopeptidase, dipeptidylpeptidase IV, and alkaline phosphatase, that are also typically expressed in the enterocytes of the small intestine.20

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Papp = (dC /dt )(Vr /ACo)

(1)

where dC/dt is the cumulative flow (mg/mL/s) determined from the linear slope of the equation defining the variation in mercury concentration (corrected for dilution) versus time, Vr is the volume of the receptor compartment (apical, 1.5 mL; basal, 2 mL), A is the surface of the cell monolayer (4.67 cm2), and Co is the initial mercury concentration in the donor compartment (mg/L). The efflux ratio (Er) was calculated from eq 2

Er = Papp(basolateral−apical)/ Papp(apical−basolateral)

MATERIAL AND METHODS

(2)

Cell Monolayer Integrity and viability. During the transport assays, cell monolayer integrity was evaluated by measuring (a) TEER at various points in the study, including the start and end of the experiment, and (b) the Papp of the paracellular transport marker lucifer yellow (LY), added at a concentration of 100 μM to the apical compartment in the control wells and the wells treated with mercury species. The fluorescence of the LY transported to the basolateral side was measured with a fluorescence microplate reader (PolarSTAR OPTIMA, BMG-Labtech, Germany) at excitation/emission wavelengths of 485/520 nm. To evaluate possible interactions of LY with the uptake and transport of mercury species, parallel experiments were performed with and without the paracellular marker, which demonstrated the absence of interference. At the end of each assay, the number of viable cells was quantified using the trypan blue-exclusion technique (trypan blue solution, 0.4%, Sigma). In all assays, the transport and accumulation values were corrected for the number of viable cells determined in this way, and they were expressed as nanograms of mercury per 106 cells. Study of Efflux. CH3Hg efflux assays were performed in transwell systems at pH 7.4. A solution of 1 mg/L (4.6 μM) of CH3Hg in HBSS-HEPES (1.5 mL) was added to the apical compartment, and HBSS-HEPES (2 mL) was added to the basolateral compartment. After incubation (37 °C, 60 min), some of the wells were used to evaluate the cellular mercury content. In the rest of the wells, the apical and basolateral media were eliminated and replaced by HBSS-HEPES without the addition of mercury followed by the study of the efflux for 240 min. At the specified time points (5, 15, 30, 60, 90, 120, 150, 180, 210, and 240 min), the apical and basal media were eliminated and replaced by the same volume of HBSS-HEPES without the addition of mercury. At the end of the efflux assay, the cells were recovered by trypsinization. Mercury was quantified in the apical and basolateral media as well as in the cells. Study of the Kinetic Parameters. The assays were performed in the A−B and B−A directions. The concentrations of CH3Hg used in the A−B direction tests were 0.07, 0.16, 0.23, 0.27, and 0.33 mg/L

Cell Culture. The human colon carcinoma cell line Caco-2 was obtained from the European Collection of Cell Cultures (ECACC, no. 86010202, Salisbury, UK). The cells were maintained in 75 cm2 flasks, to which 10 mL of Dulbecco’s modified Eagle medium (DMEM) containing 4.5 g/L glucose and 0.87 g/L glutamine supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES (N-2-hydroxyethylpiperazineN′-2-ethanesulfonic acid), 100 U/mL of penicillin, 0.1 mg/mL of streptomycin, and 0.0025 mg/L of amphotericin B was added (DMEMc). The cells were incubated at 37 °C in an atmosphere with 95% relative humidity and a CO2 flow of 5%. The medium was changed every 2 to 3 days. When the cell monolayer reached 80% confluence, the cells were detached with a solution of trypsin (0.5 g/L) and EDTA (ethylenediaminetetraacetic acid, 0.22 g/L) and reseeded at a density of 5−6.5 × 104 cells/cm2. The assays were performed with cultures between passages 27 and 42. All of the reagents used were obtained from PAA Laboratories GmbH (Labclinic, Spain). The transport assays were carried out in 6-well plates with polyester membrane inserts (24 mm diameter, pore size 0.4 μm, Transwell, Costar Corporation, Sigma, Spain). In this system, the cells are seeded onto the porous membrane of the insert that separates the well into two compartments: apical (upper) and basolateral (lower). Cells were seeded (6.5 × 104 cells/cm2) onto the inserts with the addition of 1.5 mL of DMEMc to the apical chamber and 2 mL of DMEMc to the basolateral chamber. The cells were incubated at 37 °C with 5% CO2 and 95% relative humidity, with a change of medium every 2 to 3 days until cell differentiation was attained (12−14 days postseeding). To evaluate the evolution of the monolayers during cell differentiation in the two-compartment system, the transepithelial electrical resistance (TEER) was measured using a Millicell-ERS volt-ohm meter (Millipore Corporation, Spain). The cell monolayer was considered completely formed when values of ≥250 Ω cm2 were recorded. Transport Assays and Calculation of Apparent Permeability. The transport assays were carried out in transwell systems at pH 7.2 255

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(equivalent to 0.32, 0.74, 1.06, 1.24, and 1.52 μM, respectively), whereas in the B−A direction, the concentrations were 0.10, 0.13, 0.23, 0.25, 0.30, 0.53, and 1 mg/L (equivalent to 0.46, 0.59, 1.06, 1.15, 1.38, 2.43, and 4.63 μM, respectively). After treatments (60 min, pH 7.4, 37 °C), apical and basal media and cells were recovered, and mercury quantification was carried out. The values corresponding to the mercury content standardized for the number of cells were used to calculate the kinetic parameters by fitting the data to eq 3 using nonlinear regression analysis (SigmaPlot version 12.0).

J = (Jmax C)/(K m + C) + KdC

((CH3)3N(Cl)CH2CH2OH, Sigma). The osmolarity of the media was measured using a freezing-point osmometer (Automatic MicroOsmometer type 15 Löser, Lö ser Messtechnik, Germany). All prepared media had an osmolarity of 310 ± 10 mOsm/kg. The CH3Hg treatment (0.6 mg/L, 2.8 μM) was prepared in control medium or in medium without NaCl and was added to the apical compartment (1.5 mL). Control medium (2 mL) was added to the basolateral compartment. After 120 min of exposure (pH 7.4, 37 °C), the apical and basal media and cells were recovered, and mercury quantification was carried out. Effect of the Presence of Cysteine Forms on CH3Hg Transport and Accumulation. Following the protocol of Zalups and Ahmad,8 5 μM L-cysteine (L-Cys, Merck), homocysteine (Hcys, Sigma), or N-acetyl-L-cysteine (NAC, Sigma) was kept in contact with 2.5 μM of CH3Hg (equivalent to 0.5 mg/L) in HBSS-HEPES medium for 10 min at room temperature. A transport assay was then carried out in the A−B direction by adding 1.5 mL of the mixtures of CH3Hg−cysteine forms to the apical compartment and 2 mL of HBSS-HEPES to the basolateral compartment. After exposure (120 min, pH 7.4, 37 °C), the apical and basal media and cells were recovered, and mercury quantification was carried out. In addition, the effect of increasing concentrations of L-Cys upon CH3Hg transport (0.8 mg/L, 4 μM) was evaluated. The concentrations of L-Cys used were 2.5, 20, and 50 μM. After 120 min of exposure at pH 7.4 and 37 °C, the apical and basal media were collected along with the cells for the evaluation of mercury content. Effect of Inhibitors on CH3Hg Transport and Accumulation. All of the applied treatments are described in Table 1. The inhibition

(3)

where J (pmol/min/cm /10 cells) is the flux normalized to unit surface area, Km (μM) is a constant equivalent to a Michaelis−Menten constant, Jmax is the maximal flux for the saturable term (pmol/min/ cm2/106 cells), Kd is the constant for the nonsaturable term (μL/min/ cm2/106 cells), and C is the concentration in the donor side (μM). Effect of Albumin on CH3Hg Permeability. This study was carried out in transwell systems (pH 7.4, 37 °C, 120 min) in the A−B direction. A solution of 0.3 mg/L (1.4 μM) of CH3Hg in HBSSHEPES (1.5 mL) was added to the apical compartment, and 4% (w/v) bovine serum albumin (BSA, Sigma) in HBSS-HEPES (2 mL) was added to the basolateral side. After the exposure, Papp and the Hg contents in the cellular lysate were determined as previously described. Paracellular Transport. This study was carried out in in the A−B direction (pH 7.4, 37 °C, 120 min) after incubating the cells for 5 min with 5 mM EDTA (Sigma) in PBS without Ca2+ and Mg2+ (PAA), which was added to both compartments. The pretreatment was subsequently removed, the monolayer was washed with PBS without Ca2+ and Mg2+, 1.5 mL of HBSS medium without Ca2+ and Mg2+ (PAA) containing CH3Hg (1 mg/L, 4.6 μM) and LY (100 μM) was added to the apical compartment, and 2 mL of HBSS-HEPES was added to the basolateral compartment. Papp of CH3Hg and LY was evaluated using eq 1. Participation of Energy-Dependent Transport. Two approaches were used to evaluate energy dependency. Initially, the effect of temperature on transport was analyzed by evaluating the A−B permeability of CH3Hg (1 mg/L, 4.6 μM, pH 7.4, 120 min) at 17 and 2 °C, and the results were compared with the Papp obtained at 37 °C. The Papp values at the different temperatures in turn allowed us to calculate the energy of permeation (Ep) based on the Arrhenius equation 2

Papp = P0e−E p/ RT

6

Table 1. Conditions Used in the Assay of CH3Hg Transport Inhibition inhibitor/ substrate

concentration (μM)

rifamycin BCHa L-phenylalanine L-arginine

100 100 1000 1000

a

inhibited transporter OATPs L system amino acid transporters (B0,+, L, y+L) amino acid transporters (B0,+, b0,+, L, y+L)

BCH, aminobicyclo-(2,2,1)-heptane-2-carboxylic acid.

assays were carried out in the A−B direction at pH 7.4 by adding the inhibitor or substrate prepared in HBSS-HEPES to the apical compartment. The cell monolayer was preincubated for 60 min with the corresponding inhibitor or substrate (rifamycin SV sodium salt (Fluka Chemika Biochemika, Spain), L-phenylalanine (Merck), Lmethionine (Merck), or BCH (aminobicyclo-(2,2,1)-heptane-2carboxylic acid, Sigma)). Then, without eliminating the inhibitor or substrate, we added the standard solutions of CH3Hg (0.4 mg/L, 1.84 μM) prepared in HBSS-HEPES. After the assay period (60 min), the apical and basal media were removed, the cells were recovered, and mercury content was quantified. Evaluation of the Participation of Passive Transcellular Transport. These experiments were performed using parallel artificial membrane permeability assays (PAMPA) with Multiscreen filter 96well plates (Millipore, Spain). A 1% (w/v) solution of lecithin (Sigma) in dodecane (Merck) was prepared, sonicated to ensure complete dissolution, and added carefully to each donor plate well (5 μL). Immediately after application of the lecithin/dodecane solution, 150 μL of CH3Hg (50 and 100 μM) prepared in 5% (v/v) dimethyl sulfoxide (DMSO, Sigma) in PBS at pH 7.4 was added to each well of the donor plate. As a control, verapamil hydrochloride (Sigma) at concentrations of 100 and 500 μM was used. Receiver plates with 300 μL of 5% DMSO in PBS were then coupled with the donor plates, and the resulting plate assemblies were incubated at room temperature without agitation for 16 h in a sealed container with wet paper towels to avoid evaporation. After incubation, samples from the donor and receptor plates were recovered for analysis of the mercury contents. The concentrations of

(4)

where Papp represents the apparent permeability coefficient, P0 is a preexponential factor, R is the gas constant, and T is the absolute temperature in Kelvin. By plotting the logarithm of Papp versus 1/T, Ep was determined from the slope of the linear fitting. To elucidate the participation of energy-dependent transport further, cells were preincubated with the metabolic inhibitor sodium azide (5 mM, Sigma) together with 2-deoxyglucose (50 mM, Sigma) for 10 min prior to CH3Hg exposure (0.8 mg/L, 4 μM, 120 min). Papp and Hg contents in the cellular lysate were evaluated as described previously. Effect of the Concentration of H+ on CH3Hg Permeability and Accumulation. The study was carried out in the A−B direction by varying the pH of the apical medium (5.5) and maintaining pH 7.4 in the basolateral medium. The pH value of 5.5 was obtained by adding 20 mM of 2-N-morpholineethanesulfonic acid (MES, Sigma) to the HBSS medium (HBSS-MES). A solution of CH3Hg (1.5 mL, 0.6 mg/mL, 2.8 μM) prepared in HBSS-MES was added to the apical compartment, and 2 mL of HBSS-HEPES was added to the basolateral compartment. After 120 min, apical and basolateral media and cells were recovered, and their mercury contents were evaluated. Influence of the Na+ Ions on CH3Hg Transport and Accumulation. Control medium was prepared (10 mM HEPES, 130 mM NaCl (Panreac), 10 mM KCl (Panreac), 1 mM MgSO4 (Sigma), 5 mM glucose (Panreac), and 1 mM CaCl2 (Panreac)) together with a medium without NaCl in which the latter salt was replaced by an equimolar concentration of choline chloride 256

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verapamil in the acceptor and donor compartments were determined using a UV−vis spectrophotometer (Agilent 8453 Spectroscopy system, Agilent, Spain) at a wavelength of 279 nm. The permeability coefficient across the artificial membrane (Pam) was calculated using the equation described by Sugano et al.22

Pam = − 2.303(VdnVac/Vdn + Vac)(1/St ) log(1 − flux %/100) (5)

flux % = (Cac/Cref )100

(6)

where Vdn is the volume of the donor compartment (0.15 mL), Vac is the volume of the acceptor comportment (0.3 mL), Cac is the quantity of mercury in the acceptor well at the end (ng), Cref is the quantity of CH3Hg added to the donor well (ng), S is the membrane area (0.3 cm2), and t is the incubation time (57.600 s). Statistical Analysis. All tests were performed at least in triplicate in independent cultures. The results were subjected to statistical analysis by one-factor analysis of variance (ANOVA) with the Tukey HSD posthoc multiple-comparison test or by Student’s t test (SigmaPlot version 12.0). Statistical significance was achieved for p < 0.05.

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RESULTS The concentrations used in the experiments of this article (≤5 mg/L) reflect a real situation of human exposure.23,24 In all of the assays performed, the different concentrations of mercury added to the Caco-2 cells did not affect the integrity of the cell monolayer. TEER values were not modified by more than 25% with respect to the initial values, and the Papp of LY at the end of the assay were under 2 × 10−7 cm/s. In turn, viability was over 80% for all of the concentrations assayed. These results indicate that the changes in CH3Hg transport observed upon varying the assay conditions were not due to the destructuring of the monolayer. Moreover, it should be noted that in all of the assays conducted a mass balance was performed by comparing the mercury added to the cells initially and the mercury quantified at the end of the transport assays in the apical and basal media and in the cells. The results show recoveries ranging between 82 and 99%. Calculation of the Apparent Permeability Coefficient (Papp) and Efflux Ratio (Er). Figure 1 shows cumulative CH3Hg transport over 120 min in the A−B (Figure 1A) and B−A directions (Figure 1B). Transport was seen to increase in both directions with the exposure time, with two welldifferentiated slopes, in which the first phase exhibits the slowest transport velocity. The lag in the transport of CH3Hg during the first sampling interval may be caused by the fact that the partitioning of the mercury species into the cell monolayer is a rate-limiting step.25 The increase in velocity occurred after 30 min in the A−B direction and after 60 min in the B−A direction, independently of the assayed concentration. The amount of mercury transported to the basolateral side was greater than toward the apical side for all of the time points and concentrations assayed. Thus, after 120 min of exposure, the amount of mercury transported in the A−B direction was 9−21% of the total added, whereas in the B−A direction, the amount did not exceed 7% (Table 2). Although the percentage transport in both directions increased with the concentration, the increase was not linear (Table 2). Another important observation was the high cellular contents in both directions, which was greater in the A−B direction (48−78%) than in the B−A direction (45−51%). In the same manner as that observed in the case of transport, no linear accumulation of mercury was

Figure 1. Cumulative methylmercury transepithelial transport as a function of time (pH 7.4, 37 °C). (A) Transport in the A−B direction following Caco-2 cell exposure to 1 and 5 mg/L of CH3Hg. The inset represents A−B transport in Caco-2 cells exposed to 0.1 and 0.5 mg/L of CH3Hg. (B) Transport in the B−A direction after exposure to 0.5 and 1 mg/L of CH3Hg. Values are expressed as nanograms of mercury per 106 cells (mean ± standard deviation, n = 3).

Table 2. Percentage of Mercury in Apical and Basolateral Media and Cell Monolayers after 120 min of Exposure of Caco-2 cells to CH3Hg (pH 7.4, 37 °C) in the Apical− Basolateral Direction (A−B) (0.1, 0.5, 1, and 5 mg/L) and in the Basolateral−Apical Direction (B−A) (0.5 and 1 mg/L)a percentage of mercury direction

CH3Hg (mg/L)

A−B

0.1 0.5 1 5 0.5 1

B−A

apical 47 25 20 10 49 47

± ± ± ± ± ±

6.2 0.4b 1.2b 0.9b 0.6 2.0

cellular 48 67 71 78 45 51

± ± ± ± ± ±

2.9 0.5b 8.9 3.4 1.2 0.6b

basolateral 8.9 12 21 20 4.6 6.7

± ± ± ± ± ±

0.2 0.4b 0.1b 1.1 0.4 0.2b

a

Values are expressed as a percentage with respect to the amount of CH3Hg added initially (mean ± standard deviation, n = 3). bIndicates statistically significant differences (p < 0.05) with respect to the next lower concentration.

observed with increasing concentration in either of the assayed directions. 257

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The apparent permeability coefficient evaluates the velocity with which a solute is transported through the cell monolayer. Table 3 shows the values of Papp calculated in both directions Table 3. Apparent Permeability Coefficients (Papp; cm/s) and Efflux Ratio (Er) of CH3Hg in Exposed Caco-2 Cells in the Apical−Basolateral Direction (A−B) (0.1, 0.5, 1, and 5 mg/L) and in the Basolateral−Apical Direction (B−A) (0.5 and 1 mg/L) over 120 min (pH 7.4, 37 °C)a CH3Hg (mg/L) 0.1 0.5 1 5

Papp A−B (1 × 10−6)

Papp B−A (1 × 10−6)

Er

± ± ± ±

0.86 ± 0.20 0.75 ± 0.08

0.27 ± 0.05 0.14 ± 0.01b

1.62 3.13 4.96 5.37

0.11 0.06b 0.02b 0.33b

Values are expressed as the mean ± standard deviation (n = 3). Indicates statistically significant differences (p < 0.05) with respect to the next lower concentration. a b

after 120 min of exposure to different concentrations of CH3Hg. In the A−B direction, changes in Papp were observed according to the assayed concentration, with a significant increase being observed for concentrations between 0.5 and 5 mg/L. In the B−A direction, no differences in Papp were observed between concentrations, and the values were lower than those recorded in the A−B direction. The efflux ratio (Er) values were