Correlation of in vitro cytotoxicity with paracellular permeability in Caco-2 cells

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  • ityo-2

    , F

    and A

    ica, N

    Received 27 January 2005; accepted 22 March 2005

    electrical resistance (TEER)

    cedures and their use in validation study designs (ICC-

    VAM publications 01-4499 and 01-4500, 2001). In its

    trointestinal absorption, among other methods, to

    improve the ability of in vitro cytotoxicity assays to pre-

    dict rodent LD50 values, or any in vivo toxic eects.

    Consequently, we sought to apply a cell culture model,previously used for pharmacokinetic evaluation of in

    .

    * Corresponding author. Tel.: +1 718 990 2640; fax: +1 718 990 1877.

    E-mail address: barilef@stjohns.edu (F.A. Barile).

    Toxicology in Vitro 10887-2333/$ - see front matter 2005 Elsevier Ltd. All rights reserved1. Introduction

    In a recent workshop, the Interagency Coordinating

    Committee on the Validation of Alternative Methods(ICCVAM) and the National Toxicology Program

    (NTP) Interagency Center for the Evaluation of Alter-

    native Toxicological Methods (NICEATM) forwarded

    recommendations on the development of candidate pro-

    report, the workshop summarized the validation status

    and current potential uses of in vitro methods as predic-

    tors of acute in vivo toxicity. Among the major recom-

    mendations, the report concluded that in order tofurther reduce the use of animals in acute lethality as-

    says, it is necessary to develop simple predictive models

    for human acute cytotoxicity (AC). The group encour-

    aged the optimization of simple systems that mimic gas-Abstract

    This in vitro study aims to develop a cell culture model that compares paracellular permeability (PP) with acute cytotoxicity

    (AC). Caco-2 cells were seeded in 96-well plates and on polycarbonate lter inserts. Conuent monolayers were exposed to increas-

    ing concentrations of 20 reference chemicals for 24-h and 72-h. Cytotoxicity was determined using MTT and NRU cell viability

    assays in 96-well plates. PP was measured using transepithelial electrical resistance (TEER) measurements, as well as passage of luci-

    fer yellow (LY), [3H]-mannitol (both low mw indicators), and FITC-dextran (higher mw indicator) in culture inserts. Inhibitory con-

    centrations 50% (IC50s) suggest that there were good correlations between 24-h and 72-h exposures. NRU IC50 values correlated

    better with TEER, which is consistent with the Registry of Cytotoxicity (RC; ICCVAM) database report. Both cell viability assays

    indicate that cytotoxicity occurs before TEER is compromised. In addition, 24-h and 72-h NRU assays, and 72-h TEER measure-

    ments, displayed the highest correlations with established rodent LD50s. PP experiments showed that passive paracellular transport

    of the tight junction markers, especially [3H]-mannitol, correlates with the IC50s determined with the viability assays and TEER

    measurements. Our AC/TEER/PP model thus allows for the dierentiation between the concentrations necessary for AC and those

    needed to interfere with PP. We propose that the in vitro AC, TEER and PP results be used to compute a formula which can nor-

    malize and improve the predictive ability of in vitro acute cytotoxicity assays for in vivo lethality.

    2005 Elsevier Ltd. All rights reserved.

    Keywords: In vitro cytotoxicity; Paracellular permeability (PP); Caco-2; Neutral red uptake assay (NRU); MTT viability assay; TransepithelialCorrelation of in vitro cytotoxicin Cac

    Roula Konsoula

    Department of Pharmaceutical Sciences, College of Pharmacy

    Parkway, Jamadoi:10.1016/j.tiv.2005.03.006with paracellular permeabilitycells

    rank A. Barile *

    llied Health Professions, St. Johns University, 8000 Utopia

    Y 11439, USA

    www.elsevier.com/locate/toxinvit

    9 (2005) 675684

  • groups consist of cells in media (minus chemical) which

    are processed identically and incubated simultaneously

    icologvitro absorption, as a toxicokinetic tool for predicting

    acute systemic toxicity. The intention is to correlate

    well-documented AC methods with paracellular perme-

    ability (PP), using a reference set of chemicals.

    Several investigators have recommended a strategy to

    reduce the number of animals required for acute oraltoxicity testing by using in vitro cytotoxicity data to

    determine the starting dose for in vivo testing (Halle

    et al., 2000; Spielmann et al., 1999). Also known as

    the ZEBET approach, this method is based on the stan-

    dard regression between mean IC50s and corresponding

    acute oral LD50 data included in the Registry of Cyto-

    toxicity (RC; ICCVAM publication 01-4499, 2001).

    [The ZEBET database, of which the RC is part of, pro-vides acute oral LD50 data from rats and mice and aver-

    age IC50 values of chemicals and drugs from a variety of

    in vitro cytotoxicity assays and cell types, based on 347

    chemicals. The LD50 data is derived from the NIOSH

    Registry of Toxic Eects of Chemical Substances

    (RTECS)]. The calculated regression could then be used

    to estimate the LD50 value of a new compound as the in

    vivo starting dose of a study. Others, however, have rec-ognized that the application of this technique is limited

    by the lack of information on in vitro models for gastro-

    intestinal uptake, as well as bloodbrain barrier passage

    and biotransformation (Curren et al., 1998). Concerning

    the former, monolayers of intestinal and colonic epithe-

    lial cells have, in fact, been used for many years as cell

    culture models for detecting transepithelial transport

    of drugs, and other intestinal responses to xenobiotics(Carriere et al., 2001). The connection between the eect

    of chemicals on in vitro models for PP and AC testing,

    however, has not been solidied.

    Artursson (1990) and Artursson and Karlsson (1991)

    rst described the calculation of a good correlation be-

    tween oral drug absorption in humans and drug perme-

    ability coecients in Caco-2 cells. They went on to

    conclude that paracellular absorption in humans canbe studied mechanistically in in vitro models of rat intes-

    tinal segments and Caco-2 cells (Artursson et al., 2001).

    While studies have focused on the biopharmaceutical

    development of Caco-2 cell culture models as eective

    drug delivery systems and high-throughput screening

    methods for a variety of compounds (Biganzoli et al.,

    1999; Yamashita et al., 2000, 2002), only a few have

    hinted at the relationship between paracellular transportand cell viability. For instance, Duizer et al. (1998) re-

    port that enhancement of transport of palmitoyl carni-

    tine chloride, a commonly used absorption enhancer,

    in Caco-2 cells, correlated with reduced cell viability.

    Karlsson et al. (1999) conrmed these ndings by sug-

    gesting that a pronounced disruption of the tight junc-

    tion barrier is required for ecient enhancement of

    paracellular intestinal drug transport.Together with well documented in vitro AC methods,

    676 R. Konsoula, F.A. Barile / Toxa system for identifying the eects of chemicals on PPas treated groups. Incubation medium consists of

    DMEM-10 supplemented as above. In the last hour of

    incubation, 10-ll MTT solution (5 mg/ml in DMEM) isadded to each well. The medium is then replaced with100-ll dimethylsulfoxide (DMSO), agitated for 5 mincould yield a precise scheme for estimating in vivo toxic

    doses. The current study aims at the development of an

    in vitro test system for PP, which in combination with

    AC procedures, can improve the predictive ability of

    in vitro acute cytotoxicity assays for in vivo lethality.

    2. Materials and methods

    2.1. Cell culture

    Cell culture supplies were obtained from Life Tech-

    nologies (Carlsbad, CA, USA) or VWR (Bridgeport,

    NJ, USA). Chemicals (>99.9% purity) were obtainedfrom Sigma Chemical Co., (St. Louis, MO, USA) and

    from Alfa Products (Ward Hill, MA, USA). Immortal

    human colon epithelial cells (Caco-2, HTB-37, Ameri-

    can Type Culture Collection, Rockville, MD, USA),

    passage numbers 22-40, were subcultured and seeded

    at 104 cells/cm2 in either 96-well plates or onto 12-well

    plates tted with Isopore PCF polycarbonate Millicell

    culture plate inserts (5 104 cells/insert). Cultures weregrown in Dulbeccos modied Eagles medium supple-mented with 10% fetal bovine serum (DMEM-10), 1%

    non-essential amino acids (NEAA), 1% glutamine,

    50 U/ml penicillin and 50 lg/ml streptomycin in anatmosphere of 7.5% CO2 and 100% humidity in air.

    The chemicals used in the studies were suggested by

    the Registry of Cytotoxicity (RC; Halle, 2003); they

    were selected based on the verication of the data set(RC-II), and for their validity in establishing a regres-

    sion model between oral LD50 values and single

    mammalian cell line IC50 values (ICCVAM publication

    01-4500, 2001).

    2.2. Assay procedures

    2.2.1. MTT cell viability assay

    In 96-well plates conuent monolayers of Caco-2 cells

    were achieved in 7-days and were incubated with increas-

    ing concentrations of each chemical for 24-h and 72-h

    (chemicals are listed in Table 1). The MTT assay (Mos-

    mann, 1983; Dolbeare and Vanderlaan, 1994) was mod-

    ied as previously described (Barile and Cardona, 1998;

    Schmidt et al., 2004). Briey, cells are exposed to increas-

    ing concentrations of the chemical (12 wells per concen-tration-group plus 1 control group) for 24-h or 72-h at

    37 C in an atmosphere of 7.5% CO2 in air. Control

    y in Vitro 19 (2005) 675684at 25 C, and the absorbance is read at 550 nm on the

  • R m

    72-h

    83

    009

    6

    04

    66

    009

    3

    8

    74

    22

    12

    8

    024

    27

    13

    f MT

    ; see T

    icologBioTek FL600 uorescence/absorbance plate reader.

    Cell viability is expressed as a percentage of the control

    group. The same plate contained additional wells with

    media and chemical only (without cells) and processed

    in parallel as reference blanks and to test for chemically

    Table 1

    IC50s (mmol/l) for Caco-2 cells using MTT and NRU assays, and TEE

    Chemical number Chemical compounds MTT 24-h MTT

    1 Acrylamide 13.8 6.

    2 Actinomycin 0.028 0.

    3 Antipyrine 38.0 10.

    4 Cadmium chloride 0.12 0.

    5 Cupric sulfate 1.0 0.

    6 Dimethyl formamide 193 208

    7 Doxorubicin 0.010 0.

    8 Glycerol 100 93

    9 Ibuprofen 2.2 2.

    10 Lithium sulfate 10.0 26

    11 Manganese chloride 9.6 7.

    12 Niacinamide 26 23

    13 Nickel chloride 1.78 1.

    14 q-Phenylenediamine a a

    15 Propranolol 0.41 0.

    16 Quinine HCl 0.12 0.

    17 Salicylic acid 33.8 21.

    18 Sodium dichromateb 0.33 0.

    19 Trichlorforon 0.95 0.

    20 Verapamil HCl 0.19 0.

    a q-Phenylenediamine interfered with the MTT assay.b Dihydrate salt. Statistical analysis revealed that, with the exception o

    signicantly dierent from each other (paired students t-test, P > 0.05

    R. Konsoula, F.A. Barile / Toxinduced reduction of MTT.

    2.2.2. Neutral red uptake (NRU) cell viability assay

    The NRU cytotoxicity assay was performed as de-

    scribed by Borenfreund and Puerner (1986). In 96-well

    plates conuent monolayers of Caco-2 cells are incu-

    bated with increasing concentrations of each chemical

    for 24-h and 72-h as with the MTT assay. NRU is deter-

    mined for each treatment concentration as follows:

    monolayers are incubated with test chemical for 24-h

    or 72-h, over a range of eight concentrations (includingcontrol group), at 37 C in an atmosphere of 7.5% CO2in air (100% humidity). At 21-h of incubation, the med-

    ium is aspirated, monolayers are rinsed with 150 ll PBS,and 100 ll NR medium (1:80 dilution of 0.4% stocksolution) is added for the remaining 3-h. The medium

    is aspirated, monolayers are rinsed with PBS, and

    150 ll of NR desorbing xative (ethanol/acetic acid) isadded. After shaking the cultures for 10 min, absor-bance values are read at 540 nm on the plate reader.

    Absorbance values for treatment groups are compared

    to that determined in control cultures. Control blanks

    (medium without cells containing chemical plus NR)

    are used to screen for background and to monitor pH

    changes. Relative cell viability is expressed as percent

    NRU of untreated control groups.2.2.3. Transepithelial electrical resistance (TEER)

    measurements

    For TEER measurements, Caco-2 cells are seeded

    onto 12-well plates tted with Isopore PCF polycarbon-

    ate Millicell culture plate inserts, and cultured as

    easurements, at 24-h and 72-h exposures

    NRU 24-h NRU 72-h TEER 24-h TEER 72-h

    14.3 23.2 11.5 3.6

    0.007 0.0045 0.028 0.0085

    32.3 46.8 107 75.9

    1.0 0.16 0.05 0.03

    3.35 1.0 1.8 1.7

    660 407 900 741

    0.028 0.014 0.034 0.018

    1098 821 430 707

    7.2 3.1 0.075 0.038

    14.7 9.7 145 57

    3.5 20.9 3.1 3.6

    19 13.3 189 239

    1.07 7.4 8.4 7.8

    1.24 0.50 20 11.7

    1.69 1.3 0.80 0.50

    0.12 0.44 0.80 0.30

    64 44.1 0.034 0.19

    0.18 0.14 0.05 0.01

    11.2 0.90 0.90 0.48

    1.8 0.87 0.58 0.60

    T 72-h vs. TEER 72-h (P < 0.05), none of the group comparisons were

    able 2).

    y in Vitro 19 (2005) 675684 677described by Biganzoli et al. (1999). DMEM-10, supple-

    mented as above, is added to the apical and basolateral

    chambers and replenished three times a week. Cultureswere conuent at 7-days, but maximum resistance val-

    ues (at least 1000 X cm2) were reached in intact mono-layers after 14-days. Transmembrane specic resistance

    was measured using the Millicell-ERS resistance sys-

    tem (Millipore) before and after 24-h and 72-h incuba-

    tion with test chemicals. As with the AC assays,

    blanks (inserts without cells containing media and chem-

    ical) are used to determine baseline values. Values aremeasured as X cm2, and expressed as percent TEER ofuntreated control groups.

    For all assays, dosage range-nding experiments were

    performed. The IC50s were extrapolated from concen-

    tration-eect curves using linear regression analysis.

    When the IC50 was not bracketed in the initial dosage

    range used for the chemical, the experiments were re-

    peated and the concentrations adjusted as necessary.After the determination of the IC50, each experiment

    was repeated at least two more times. Values in gures

    are expressed as percentages of control groups. Also,

    AC assays and PP assays were performed during the

    same passage numbers to prevent further dierentiation

    of Caco-2 cells, thus maintaining the cultures at an early

    stage of dierentiation. In addition, PP experiments

  • cepts, and the t-statistic (two-tailed paired students t-

    icologwere initiated when absolute TEER measurements

    reached 1000 X cm2 (2 weeks after seeding). This con-sistent manipulation also kept the cultures as high-resis-

    tant variants.

    2.2.4. Paracellular permeability (PP) assays

    For PP assays, Caco-2 cells were seeded onto 12-well

    plates tted with Isopore PCF polycarbonate Millicell

    culture plate inserts, as described above for TEER mea-

    surements. Cultures were incubated with the test chem-

    icals for 24-h and indicators were introduced in the last

    90 min of incubation. Low and high molecular weight

    indicators were used to determine the eect of test chem-

    icals on PP. [3H]-mannitol (mw 182) has low lipophilic-ity; FITC-dextran (mw 40 K50 K) and lucifer yellow

    (LY, mw 450) are more hydrophobic; all permeate

    paracellularly.

    Mannitol (0.1% w/v) is dissolved in DMEM plus

    HEPES, spiked with [3H]-mannitol (30 Ci/mM), and

    added to the apical chamber to a nal concentration

    of 1 mCi/l (Liu et al., 1999). Simultaneously, cold-

    DMEM (without radioactivity) is dispensed into thebasolateral chamber. At end of the incubation period,

    passage of radiolabeled marker is determined by dissolv-

    ing an aliquot of the basolateral medium in Budget-

    solve and measuring the cpm by liquid scintillation

    (Beckman LS5801 counter). Blanks (inserts without

    cells) and control cultures (minus chemical) are moni-

    tored simultaneously. Samples are also taken from the

    apical solutions to measure the initial donor concentra-tions. Background radioactivity is determined using

    DMEM, and dpm is calculated based on the instru-

    ments counting eciency for [3H] (45%).Fluorescent marker molecules are used at concentra-

    tions of 1 mg/ml in DMEM and applied to the apical

    chamber. At the end of the exposure period, the basolat-

    eral medium is collected and the concentration of LY

    and FITC-dextran is determined by measuring uores-cence intensity with the BioTek FL600 uorescence/

    absorbance microplate reader. Blanks (inserts without

    cells) are also monitored simultaneously. The excitation

    and emission wavelengths for LY are 430 nm and

    540 nm, respectively, and for FITC-dextran, 487 nm

    and 518 nm, respectively. As with the above assays, uo-

    rescence values for treatment groups are compared to

    that determined in control cell cultures. Relative cellpermeability is expressed as a percent of untreated con-

    trol groups.

    2.3. Solubilization or incompatibility of chemicals

    Most of the experimental problems involved the sol-

    ubilization or miscibility of some of the chemicals with

    the media. Solid chemicals that were insoluble in waterpresented with dissolution problems, especially at the

    678 R. Konsoula, F.A. Barile / Toxhigher dosage levels. The solubility of these chemicalstest with the more stringent equal variances assumption)

    are calculated for all the chemicals in a particular assay

    as previously described, using Microsoft Excel (Ekwall

    and Barile, 1994).In addition, the regressions calculated for the AC

    studies are compared with the RC regression for the

    20 chemicals tested, to contrast our model cytotoxicity

    test with the RC prediction model. The RC regression

    equation is based on the following formula:

    logLD50 0.435 logIC50 0.625where r = 0.67 for 347 chemicals in the RC database

    (Halle et al., 2000) and 0.97 for 11 of the 20 chemicals

    (ICCVAM publication 01-4499, 2001) used in this study.

    If the regression line obtained with our model parallels

    the RC regression and is within log5 interval, then

    the test is considered suitable to generate IC50 data to

    use with the RC regression for estimating starting doses.

    3. Results

    3.1. Comparison of cytotoxicity viability data and TEER

    measurements

    Table 1 shows the IC50 data generated from the AC

    and TEER studies. The data summarizes the experi-ments for 24-h and 72-h. Cytotoxicity was determined

    using MTT and NRU cell viability assays (AC studies)

    on conuent Caco-2 cells in 96-well plates, and TEER

    was measured in conuent monolayers on Millipore

    polycarbonate lter membranes, as described above.

    IC50s were calculated from regression analyses (not

    shown). Table 2 summarizes the statistical analysis of

    the data in Table 1. MTT, NRU, and TEER assays werecompared to each other at corresponding times (24-h

    and 72-h). The coecient of determination (R2) andwas improved by the addition of a solubilizer to the

    incubating medium as follows: salicylic acid and ibupro-

    fen1 N NaOH; propranolol, verapamil, doxorubicin,

    actinomycin1% DMSO; quinine sulfate1% ethanol.

    In separate experiments we determined that none of the

    additives inuenced control group cytotoxicity or para-cellular permeability of [3H]-mannitol.

    q-Phenylenediamine was excluded from the MTTassay data because of its propensity for reducing the sub-

    strate and interfering with absorbance measurements.

    2.4. Statistical analysis

    IC50 values are extrapolated from concentration-eect curves using linear regression analysis for the aver-

    age of at least three experiments. The coecient of

    determination (R2), regression analysis, slopes, inter-

    y in Vitro 19 (2005) 675684slope (m) of the comparisons are indicated. R2 measures

  • the degree of correlation between the sets of data, while

    the slope is an indication of the deviation of the plot of

    experimental values from a 1:1 (mM:mM) relationship.

    With the exception of MTT 72-h vs. TEER 72-h, none

    of the sets of data were signicantly dierent from each

    other (P > 0.05, Tables 1 and 2).

    The regressions indicate that there was a high corre-

    lines suggest that the 24-h assays, in general, are the

    more sensitive procedures (in the calculation of slope

    on log scale, when m is less than 1.0, the line is shifted

    to the right and the y-values are lower than correspond-

    ing x-values). High correlations were also obtained with

    the 72-h MTT and NRU assays vs. 72-h TEER(R2 = 0.84 and 0.82, respectively). Both assays were also

    more sensitive than the TEER measurements (m = 0.20

    and 0.80, respectively). Our AC/TEER analysis thus al-

    lows for the dierentiation between the concentrations

    necessary for AC and those needed to interfere with

    TEER. These results suggest that the MTT assay is a

    more sensitive indicator of chemical exposure than

    NRU or TEER measurements (m = 0.20, 0.20, 0.13,0.18; Table 2). In fact, the model thus far reveals that

    at equivalent IC50s, mitochondrial activity is more likely

    to be altered before paracellular permeability is

    compromised.

    3.2. Comparison of cytotoxicity viability data and RC

    database

    Table 3 compares the IC50s obtained from the cyto-

    toxicity assays and resistance measurements against

    Table 2

    Statistical comparison of IC50 data from Table 1 for Caco-2 cells using

    MTT and NRU assays, and TEER measurements at 24-h and 72-h

    exposures for the 20 reference chemicals according to regression

    IC50 Data (X vs. Y) Number of chemicals R2 m

    NRU 24-h vs. NRU 72-h 20 0.99 0.71

    MTT 24-h vs. MTT 72-h 19 0.96 0.87

    MTT 24-h vs. TEER 24-h 19 0.95 0.20

    TEER 24-h vs. TEER 72-h 20 0.89 0.91

    MTT 72-h vs. TEER 72-ha 19 0.84 0.20

    NRU 72-h vs. TEER 72-h 20 0.82 0.80

    MTT 24-h vs. NRU 24-h 19 0.69 0.13

    NRU 24-h vs. TEER 24-h 20 0.62 1.00

    MTT 72-h vs. NRU 72-h 19 0.53 0.18

    a With the exception of MTT 72-h vs. TEER 72-h (P < 0.05), none of

    the group comparisons were signicantly dierent from each other

    (paired students t-test, P > 0.05). R2 = coecient of determination ofregression analysis; m = slope of line of best t.

    50s f

    data

    0 (mm

    .61

    R. Konsoula, F.A. Barile / Toxicology in Vitro 19 (2005) 675684 679lation between 24-h and 72-h intra-assay cytotoxicityfor NRU (R2 = 0.99), MTT (R2 = 0.96), and TEER

    (R2 = 0.89; Table 2). There was also a high correlation

    between the MTT 24-h and TEER 24-h (R2 = 0.95).

    Although there were no signicant dierences among

    all assays (with one exception), the atter slopes of the

    Table 3

    IC50 data for in vitro assays with the highest correlations with the RC LD

    the same chemicals

    Chemical

    number

    Chemical

    compounds

    RC database

    LD50 (mmol/kg)a

    RC

    IC5

    1 Acrylamide 2.39 12 Actinomycin 0.0057 0.0000

    3 Antipyrine 9.56 11.6

    4 Cadmium chloride 0.48 0.0064

    5 Cupric sulfate 1.2 0.33

    6 Dimethyl formamide 38.3 114

    7 Doxorubicin 1.2 0.0003

    8 Glycerol 137 624.0

    9 Ibuprofen 4.89 0.52

    10 Lithium sulfate 10.8 34

    11 Manganese chloride 7.5 0.13

    12 Niacinamide 28.7 44

    13 Nickel chloride 0.81 0.27

    14 q-Phenylenediamine 0.74 0.0515 Propranolol 1.59 0.12

    16 Quinine HCl 1.72 0.075

    17 Salicylic acid 6.45 3.380

    18 Sodium dichromateb 0.19 0.0009

    19 Trichlorforon 1.75 0.27

    20 Verapamil HCl 0.22 0.10

    a Values obtained from ICCVAM guidance document Registry of Cytotoxb Dihydrate salt.the LD50s for the same representative chemicals from

    the RC database. Statistical analysis of the data is pre-

    sented in Table 4. The regressions indicate that the RC

    IC50s and both 24-h and 72-h NRU assays have the

    highest correlations to the RC LD50s (R2 = 0.97, 0.92,

    and 0.88, respectively). In addition, the 72-h TEER

    or the 20 reference chemicals, as well as IC50s from the RC database for

    base

    ol/l)aNRU 72-h

    IC50 (mmol/l)

    NRU 24-h

    IC50 (mmol/l)

    TEER 72-h

    IC50 (mmol/l)

    23.2 14.3 3.6

    081 0.0045 0.007 0.0085

    46.8 32.3 75.9

    0.16 1.0 0.03

    1.0 3.35 1.7

    407 660 741

    3 0.014 0.028 0.018

    821 1098 707

    3.1 7.2 0.038

    9.7 14.7 57

    20.9 3.5 3.6

    13.3 19 239

    7.4 1.07 7.8

    0.5 1.24 11.7

    1.3 1.69 0.50

    0.44 0.12 0.30

    44.1 64 0.19

    3 0.14 0.18 0.01

    0.90 11.2 0.48

    0.87 1.8 0.60

    icity (RC) database, 2001.

  • measurements also rank with a good correlation with

    the LD50s (R2 = 0.70). The low values of the slopes indi-

    cate that in all cases, LD50s are more sensitive than all

    IC50 values. Only the 24-h MTT assay approaches the

    sensitivity of the LD50 values (m = 0.46), although the

    data does not correlate well (R2 = 0.42). It was not unex-

    pected, therefore, that the 72-h NRU protocol would

    demonstrate comparable correlation with RC LD50data, as the RC IC50s, since the RC IC50s are based

    on the NRU assay. In fact, our NRU IC50s in Caco-2

    cells mirrored the RC NRU correlation performed in

    BALB/c 3T3 mouse broblasts for 72-h (Table 4).

    Fig. 1 compares IC50s derived from NRU and TEER

    assays with IC50s for the same representative chemicals

    from the RC database. The regression plots (ac) show

    good correlations, suggesting that AC and TEER mea-surements together are consistent with the RC IC50s.

    Although the correlations of the three procedures with

    the RC IC50s have high correlations and parallel the

    LD50s (Table 4), the slopes of the lines are systematically

    higher (the data is shifted to the left, rendering the slopes

    greater than 1.0). Consequently, on a logarithmic scale,

    this suggests that the IC50 concentrations for the indi-

    cated assays are slightly higher than those reported inthe RC database.

    3.3. Comparison of PP data and TEER measurements

    Figs. 24 illustrate the data obtained from PP exper-

    iments using passive paracellular transport markers. The

    concentrations used are based on the IC50s determined

    for the TEER experiments (Table 1) as well as the upperand lower limits (data not shown). Fig. 2 groups four

    representative chemicals with highest transport of [3H]-

    mannitol at IC50s determined for the viability assays.

    This gure illustrates that paracellular transport of

    [3H]-mannitol, as a tight junction marker, more closely

    Table 4

    Statistical comparison of IC50 data from Table 3 with LD50 data from

    the RC database for the 20 reference chemicals according to regression

    RC LD50a data (X)

    vs. IC50s (Y)

    Number of

    chemicals

    R2 m b

    RC LD50sa vs. RC IC50s

    a 20 0.97 0.22 3.7

    vs. NRU 72-h 20 0.92 0.15 2.3

    vs. NRU 24-h 20 0.88 0.10 2.7

    vs. TEER 72-h 20 0.70 0.12 2.0

    vs. MTT 24-h 19 0.42 0.46 3.3

    vs. TEER 24-h 20 0.37 0.09 4.8

    vs. MTT 72-h 19 0.34 0.37 5.7

    a From the Registry of Cytotoxicity (RC) database (ICCVAM

    guidance document, 2001). None of the group comparisons were sig-

    nicantly dierent from each other (paired students t-test, P > 0.05).R2 = correlation coecient of regression analysis; m = slope of line of

    best t; b = y-intercept, used to normalize the data (see Section 4).

    680 R. Konsoula, F.A. Barile / Toxicology in Vitro 19 (2005) 675684Fig. 1. NRU and TEER IC50s in Caco-2 cells vs. RC IC50s. Graphs of (a) 24

    IC50s obtained for the same chemicals from the RC database. The regression

    theoretical 1:1 correlation.-h NRU, (b) 72-h NRU, and (c) 72-h TEER IC50s for Caco-2 cells vs.

    s (R2) of the solid lines are indicated. The dotted line ( ) represents a

  • Fig. 2. Eect of (a) acrylamide, (b) manganese chloride, (c) glycerol, and (d) q-phenylenediamine on paracellular transport in Caco-2 cells.Concentrations correspond to IC50s generated in the TEER studies. Scale for % of control for PP markers (bars) are on left axis; for TEER

    measurements (solid line), scale is on right axis. All control values are set at 100%. FITC = uorescein isothiocyanate-dextran, LY = lucifer yellow,3H = [3H]-mannitol, TEER = transepithelial electrical resistance.

    Fig. 3. Eect of (a) niacinamide, (b) doxorubicin, (c) dimethylformamide, and (d) lithium sulfate on paracellular transport in Caco-2 cells.

    Concentrations correspond to IC50s generated in the TEER studies. Scale for % of control for PP markers (bars) are on left axis; for TEER

    measurements (solid line), scale is on right axis. All control values are set at 100%. FITC = uorescein isothiocyanate-dextran, LY = lucifer yellow,3H = [3H]-mannitol, TEER = transepithelial electrical resistance.

    R. Konsoula, F.A. Barile / Toxicology in Vitro 19 (2005) 675684 681

  • d (d)

    l for

    oresc

    682 R. Konsoula, F.A. Barile / Toxicology in Vitro 19 (2005) 675684Fig. 4. Eect of (a) cadmium chloride, (b) antipyrine, (c) quinine, an

    correspond to IC50s generated in the TEER studies. Scale for % of contro

    scale is on right axis. All control values are set at 100%. FITC = uparallels the fall in TEER than LY or FITC-dextran.

    Fig. 3 groups four representative chemicals that show

    an increase in PP of all tight junction markers more uni-

    formly as TEER decreases. Fig. 4 shows chemicals that

    inuence PP of FITC-dextran as well as [3H]-mannitolas TEER decreases. In every instance, transport of

    [3H]-mannitol to the basolateral surface correlates better

    than FITC with a decrease in resistance [Data from ini-

    tial experiments using [14C]-mannitol showed no dier-

    ences from [3H]-mannitol]. PP data for the rest of the

    20 chemicals is not shown but mimics the graphs in

    Fig. 2.

    4. Discussion

    Currently available AC methods are limited by the

    inadequate availability of in vitro models for paracellu-

    lar or transcellular transport, bloodbrain barrier pas-

    sage, and biotransformation. Recently, ICCVAM and

    the National Toxicology Program (NTP) NICEATMconvened a workshop to address several specic objec-

    tives: 1. review the status of in vitro methods for assess-

    ing acute systemic toxicity; 2. recommend candidate

    methods for further validation studies, and; 3. identify

    reference chemicals for use in the in vitro methods. Only

    TEER = transepithelial electrical resistance.verapamil on paracellular transport in Caco-2 cells. Concentrations

    PP markers (bars) are on left axis; for TEER measurements (solid line),

    ein isothiocyanate-dextran, LY = lucifer yellow, 3H = [3H]-mannitol,a few groups have attempted to deviate from the tradi-

    tional investigations of paracellular and transcellular

    transport on Caco-2 cells and incorporate cytotoxicity

    testing procedures. Chao et al. (1998) have shown that

    long-chain acylcarnitines increase intestinal absorptionof small hydrophilic compounds through the paracellu-

    lar pathway by dilating paracellular spaces in Caco-2

    monolayers. By comparing the potency index of a novel

    absorption enhancer with palmitoyl carnitine, Liu et al.

    (1999) show that dodecylphosphocholine is signicantly

    safer. This index is a ratio between the IC50 value,

    obtained using the MTT test, and EC50 value, the eec-

    tive concentration at which TEER drops to 50%. Duizeret al. (1999) detected a decrease in TEER with 5- to 30-

    lM cadmium chloride (CdCl2) exposure in Caco-2 cells,with a corresponding disruption of the paracellular bar-

    rier, yet without a signicant loss of viability. Boveri

    et al. (2004) report that CdCl2 toxicity can be detected

    in Caco-2 cells at very low concentrations using LDH

    release and HSP70 as toxicological endpoints. Okada

    et al. (2000) compared changes in TEER in Caco-2 cellmonolayers with cell viability using the LDH release as-

    say. They showed that a decrease in TEER, associated

    with increased transepithelial permeability, preceded

    LDH release. It is interesting to note that although the

    authors recommend the TEER method and LDH re-

  • AC/TEER/PP model thus allows for the dierentiation

    between the concentrations necessary for AC and those

    R. Konsoula, F.A. Barile / Toxicology in Vitro 19 (2005) 675684 683lease assay as suitable for detecting a variety of marine

    toxicants, the procedure may not be useful for agents

    which inhibit protein synthesis or that require incuba-

    tion times beyond 3 h. Shah et al. (2004) evaluated the

    cytotoxicity of class specic compounds (enzyme inhib-

    itors and absorption enhancers) in Caco-2 cells using avariety of permeability and viability assays, including a

    tetrazolium-based assay. Toxicity varied with time of

    incubation; correlations between absorption and viabil-

    ity, however, were not reported.

    We obtained high regression values between AC

    IC50s and TEER measurements (Table 2), indicating

    that PP varies directly with AC; i.e. as AC increases,

    transepithelial resistance decreases proportionately.According to the regression standards for the RC data-

    base, cytotoxicity is predicted by our AC/PP model.

    However, almost all of the slopes for the AC assays

    vs. TEER measurements are less than 1.0, suggesting

    that AC assays are more sensitive than TEER. Conse-

    quently, determination of R2 is not sucient for deter-

    mining sensitivity of in vitro systems. In addition,

    paracellular transport of tight junction markers, espe-cially the low-molecular weight [3H]-mannitol, corre-

    lates precisely with decreases in TEER. Thus the data

    suggests that interference with cell viability is more

    likely to occur before PP is compromised. (It is impor-

    tant to note that although some chemicals may selec-

    tively interfere with the paracellular route, overall, the

    experiments are not designed to address the mechanisms

    of acute toxicity. Nor will the protocol dierentiate be-tween passive transcellular and paracellular transport.

    The study is constructed, however, to determine the

    association between PP and cell viability. As a result,

    calculation of a standardized formula from a larger set

    of compounds would dilute any specic mechanism of

    toxicity).

    Comparison of the regressions between 72-h and 24-h

    NRU, and 72-h TEER IC50s, with RC LD50s for thesame chemicals (Table 4), indicates that the model sys-

    tem correlates well with the LD50 data. Together with

    the regression analysis, computation of the slopes of

    all plots further qualies the relationships. These calcu-

    lations (0.22, 0.15, 0.10, and 0.12; Table 4) suggest that

    in vitro AC methods correlate well with animal LD50s.

    Because the slopes are less than 1.0 however, the LD50values (mmol/kg) are lower than the in vitro IC50s(mmol/l) (slopes less than 1.0 convey a atter line of best

    t or a shift-to-the-right, both of which compute to

    higher values of X).

    PP experiments showed that passive paracellular

    transport of tight junction markers correlates with the

    IC50s determined with the TEER measurements. Since

    the IC50s for the 24-h MTT and NRU experiments were

    generally lower than the 24-h TEER measurements, andPP of tight junction markers increases systematicallyneeded to interfere with PP.

    Our study represents a systematic approach to corre-

    lating a traditional pharmacokinetic system with in vitro

    cytotoxicity testing procedures. Based on these results,

    we recommend that the AC/PP model be used together

    as part of a battery of in vitro tests to eectively screen

    for in vivo toxicity. In fact, we propose that a normal-

    ization of the results of the methods should be used forpredicting starting doses in toxicity testing. Based on the

    calculations of the averages of the slopes (m) and y-

    intercepts (b) for the best regressions obtained in these

    studies, the following formula can be used to estimate

    an LD50 or starting dose from the line of best t:

    logmmol=kg LD50 0.15logmM IC50 2.7where m = 0.15 and b = 2.7 are computed as the mean

    for the rst 4 assays with the highest regressions (Table

    4). This standardized approach for using the AC/PP

    model improves the ability to predict acute oral toxicity.

    Interestingly, knowledge of a compounds intestinal per-meability or absorption, however, may not necessarily

    be useful to further classify cytotoxicity. Comparison

    of our data with available permeability indexes for someof the compounds tested (Ingels et al., 2004; Ruell et al.,

    2003) revealed no correlation (data not shown), suggest-

    ing that permeability indexes and cytotoxicity are not

    necessarily related. It is also important to note that vari-

    ability in expression of transporter/eux systems has

    been shown to alter intracellular concentrations of toxic

    agents, and should be taken into account in the valida-

    tion of specic classes of compounds (Behrens andKissel, 2003). However, since almost all of the chemicals

    in Table 1 are not euxed compounds, P-glycoprotein

    (P-gp) or non-P-gp carriers would not aect most chem-

    icals. Furthermore, maintenance of consistent cell cul-

    ture conditions limits the inuence of carrier-mediated

    transport systems in the monolayers (Behrens and Kis-

    sel, 2003). Also, since our model incorporates 2 exposure

    times and 3 assays, a single method is not relied upon toeectively identify a toxic substance. Consequently the

    case has been repeatedly advanced that a selective bat-

    tery of validated (and normalized) tests would be to

    the advantage of any in vitro toxicology program.

    Acknowledgement

    This work was supported in part by grants from the

    NIH, NIEHS (R15 ES012170-01), USA.with a fall in TEER, it is reasonable to conclude that cel-

    lular viability is compromised before PP is aected. In

    addition, as a low molecular weight substance, [3H]-

    mannitol, is the most sensitive indicator for PP. Our

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    Correlation of in vitro cytotoxicity with paracellular permeability in Caco-2 cellsIntroductionMaterials and methodsCell cultureAssay proceduresMTT cell viability assayNeutral red uptake (NRU) cell viability assayTransepithelial electrical resistance (TEER) measurementsParacellular permeability (PP) assays

    Solubilization or incompatibility of chemicalsStatistical analysis

    ResultsComparison of cytotoxicity viability data and TEER measurementsComparison of cytotoxicity viability data and RC databaseComparison of PP data and TEER measurements

    DiscussionAcknowledgementReferences

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