Correlation of in vitro cytotoxicity with paracellular permeability in mortal rat intestinal cells

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    IEC-18, a small intestinal crypt cell line derived from the ratileac epithelium, has been used as a model to study small

    2 express TEER values that are relatively higher than monolayerscultured from the small intestine. These features may also explain

    icaintestine epithelial permeability (Duizer et al., 2002; Ma,Hollander, Bhalla, Nguyen, & Krugliak, 1991; Quaroni &Hochman, 1996). The transepithelial electrical resistance(TEER) of IEC-18 cells cultured on membrane inserts resemblesthe TEER of the rat ileum (88 cm2; Powell, 1981), whichsuggests comparable leakiness or tightness of the iliacparacellular pathways. The low TEER of IEC-18 cells, com-pared to human colon cancer cells (Caco-2), may be explainedby their crypt origin and low differentiation stage. Yet, IEC-18cells have been used to investigate the cytotoxic effects ofvariety of agents (Deitch, Haskel, Cruz, Xu, & Kvietys, 1995).

    the higher resistance to cytotoxicity, and prompts the need todevelop additional in vitro models resembling PP of the smallintestine.

    The aim of this study is to compare the acute cytotoxicresponse and permeability of IEC-18 cells in response to chemicalinsult, with previously performed studies using Caco-2. Ulti-mately, the development of a sensitive in vitro model for mea-suring PP, in combination with AC, can improve the predictiveability of in vitro AC assays for in vivo lethality.

    2. Materials and methodssmall intestine than the Caco-2. Cytotoxicity was carried out using MTT cell viability assay in 96-well plates for 24-h exposure time. PP was measuredusing TEER (membrane integrity indicator) and PPmarkers such as [3H]-D-mannitol, lucifer yellow (LY) and FITC-dextran (fluorescein-dextran) on cellsgrown on inserts.Results: The data showed that there is a high correlation (R2=0.99) betweenMTTand TEER using IEC-18 cell for 24-h exposure time.IEC-18 is as sensitive as Caco-2 for bothMTTand TEERmeasurements. Decrease in TEER is inversely proportional with increase in PP of tight junctionindicators. There is a good correlation between IC50's MTT, TEER and Registry of Cytotoxicity (RC) data. Discussion: Based on the results from theexperiments, IEC-18 can be used as an in vitro model to differentiate between concentrations needed for AC and those required for PP. 2006 Elsevier Inc. All rights reserved.

    Keywords: In vitro cytotoxicity; Paracellular permeability (PP); IEC-18; MTT viability assay; Transepithelial electrical resistance (TEER)

    1. Introduction (AC) of 20 chemicals (Konsoula & Barile, 2005). Caco-2 is animmortal cell line originating fromhuman colon. In culture, Caco-of differentiation, TEER and paracellular permeability characteristics. TheIntroduction: The rat small intestinal cell line, IEC-18, was used as an in vitro model to differentiate between acute cytotoxicity (AC) and paracellularpermeability (PP) of selected chemicals.Methods: This study compares the low resistance rat intestinal mortal cell line, IEC-18 (transepithelial electricalresistance, TEER=16010 cm2) with the high resistance human intestinal cell line, Caco-2 (TEER=900100 cm2). The two cell lines differ in state

    IEC-18 cell line is originated from the ileum and resembles more closely theOrigin

    Correlation of in vitro cytotoxicin mortal rat

    Roula Konsoula,

    St. John's University, College of Pharmacy and Allied Health Professions, Depar

    Received 19 April 200

    Abstract

    Journal of Pharmacological and ToxicologWe recently used Caco-2 monolayers as an in vitro model tocompare paracellular permeability (PP) with acute cytotoxicity

    Corresponding author. Fax: +1 718 990 1877.E-mail address: barilef@stjohns.edu (F.A. Barile).

    1056-8719/$ - see front matter 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.vascn.2006.06.001article

    with paracellular permeabilitytestinal cells

    rank A. Barile

    nt of Pharmaceutical Sciences, 8000 Utopia Parkway, Jamaica, NY 11439, USA

    ccepted 12 June 2006

    l Methods 55 (2007) 176183www.elsevier.com/locate/jpharmtox2.1. Materials

    IEC-18 cells, originating from the rat ileac epithelium, wereobtained from the American Type Culture Collection (Rockville,

  • MD, USA). Cell culture supplies were purchased from Invitrogen(Carlsbad, CA,USA) orVWR (Bridgeport, NJ, USA). Chemicalswere purchased fromSigma-Aldrich (St. Louis,MO, USA); [3H]-D-mannitol (17 Ci/mmol) was obtained from Perkin-Elmer(Boston, MA, USA).

    2.2. Cell culture

    IEC-18 (passage numbers 1625) were grown in Dulbecco'smodified Eagle's medium supplemented with 5% fetal bovineserum (DMEM-10), 1% antibioticantimycotic, 4 mM L-gluta-mine, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, in anatmosphere of 5% CO2 and 95% relative humidity at 37 C. Forviability studies, cells were seeded in 96-well plates (1104 cells/cm2) for 45 days. For transport experiments, IEC-18 cells(1105 cells/insert) were seeded on Transwell polycarbonateinserts (12-well format, 12 mm insert diameter) with a mean poresize of 0.4 m. IEC-18 cells reached confluency after 910 days.

    2.3. MTT cell viability assay

    The acute cytotoxic effects of 19 chemicals on cell viabilitywere measured using the MTT assay (Dolbeare & Vanderlaan,1994), originally described by Mosmann (1983). The tetrazo-lium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), is actively absorbed in a succinate-NADH+

    described (Konsoula & Barile, 2005; Schmidt, Cheng, Marino,Konsoula, & Barile, 2004). Briefly, IEC-18 cells were exposedto increasing concentrations of the chemical (6 wells perconcentration-group plus one control group) for 24 h. The cellswere incubated in DMEM supplemented as described above.Control groups consisted of cells in media (minus chemical)which are processed identically and incubated simultaneously astreated groups. In the last hour of incubation, 10 l MTTsolution(5 mg/ml in DMEM) is added to each well. The medium isreplaced with 100 l dimethylsulfoxide (DMSO), agitated for1 min at 25 C, and the absorbance is read at 550 nm on theBioTek FL600 fluorescence/absorbance plate reader. Cellviability is expressed as a percentage of the control group. Thesame plate contained additional wells with media and chemicalonly (without cells) and processed in parallel as referenceblanks, in order to test for chemically induced reduction ofMTT.

    2.4. Transepithelial electrical resistance (TEER)

    For TEER measurements, IEC-18 cells were seeded into 12-well plates fitted with Isopore PCF polycarbonate Millicellculture plate inserts. DMEM-10, supplemented as above, wasadded to the apical and basolateral chambers and replenishedthree times a week. Cultures were confluent at 7 days, butmaximum resistance values (160 cm2) were reached after10 days. Transmembrane specific resistance was measured using

    R. Konsoula, F.A. Barile / Journal of Pharmacologicamitochondrial-dependent reaction to yield a formazan product.The ability of the cells to reduce MTT provides an indication of

    Table 1IC50's (mmol/l) for IEC-18 and Caco-2 cells using MTT assay and TEERmeasurements at 24-h exposures

    Chemicals MTT-24 hIEC-18

    TEER-24 hIEC-18

    MTT-24 hCaco-2a

    TEER-24 hCaco-2a

    Acrylamide 10.46 8.7 13.8 11.5Actinomycin 0.033 0.012 0.028 0.028Antipyrine 26.6 79.43 38.0 107Cadmium chloride 0.077 0.16 0.12 0.05Cupric sulfate 0.28 0.30 1.0 1.8Dimethylformamide 160 676 193 900Doxorubicin 0.026 0.06 0.01 0.034Glycerol 213 954 100 430Ibuprofen 0.39 0.006 2.2 0.075Lithium sulfate 41.3 81.28 10.0 145Manganese chloride 1.75 2.04 9.6 3.1Niacinamide 23.1 159 26 189Nickel chloride 0.41 3.3 1.78 8.4Propranolol 0.15 0.77 0.41 0.80Quinine HCl 0.038 0.67 0.12 0.80Salicylic acid 6.0 0.1 33.8 0.034Sodiumdichromate b

    0.012 0.024 0.33 0.05

    Trichlorfon 0.71 1.23 0.95 0.90Verapamil HCl 0.16 0.6 0.19 0.58

    Statistical analysis revealed that, overall, the groups are significantly different(ANOVA, P

  • chemical) were used to determine baseline values (80 cm2).Values are expressed as percent of untreated control groups.

    For all assays, dosage range-finding experiments wereperformed. The IC50's were extrapolated from concentrationeffect curves using linear regression analysis. When the IC50'swas not bracketed in the initial dosage range used for the che-mical, the experiments were repeated and the concentrationsadjusted as necessary. After the determination of the IC50's, each

    2.5. Paracellular permeability (PP) of mannitol, lucifer yellowand fluorescein-dextran

    The PP studies were performed as previously described(Konsoula & Barile, 2005). Briefly, IEC-18 cells were seededonto 12-well transwell polycarbonate inserts. Cells were incubatedwith the chemicals for 24-h and indicators were introduced in thelast 90 min of incubation. Low and higher molecular weightparacellular markers were used to determine the effect of testchemicals on PP: [3H]-D-mannitol (mw=182) has low lipophili-city, whereas lucifer yellow (LY, mw=450) and fluorescein-dextran (FITC-dextran, mw=40 K50 K) are more hydrophobic.

    For radioactive experiments, D-mannitol (0.1% w/v) wasdissolved in DMEM, spiked with [3H]-D-mannitol (17 Ci/mmol), and added onto the apical side (0.5 ml) of the insert to afinal concentration of 1 mCi/L (Liu, LeCluyse, & Thakker,1999). Cold-DMEM (without radioactive mannitol) was addedin the basolateral side. At the end of the exposure period, analiquot of basolateral [3H]-D-mannitol was measured by liquidscintillation counting (Beckman LS5801 counter). Blanks(inserts without cells) and control groups (minus chemical)were monitored simultaneously. Background radioactivity wasdetermined using DMEM, and dpm was calculated based on theinstrument's counting efficiency (for [3H] 45%).

    Table 3Statistical comparison of IC50 data from Table 1 using MTT assay and TEERmeasurements for IEC-18 and Caco-2 cells, and with data from the RC database

    IC50's data (Y vs. X) R2 m

    MTT (IEC-18) vs. TEER (IEC-18) 0.99 0.23MTT (IEC-18) vs. MTT (Caco-2) 0.75 1.06TEER (IEC-18) vs. TEER (Caco-2) 0.73 0.99MTT (IEC-18) vs. RC IC50 0.77 0.36MTT (IEC-18) vs. RC LD50 0.82 1.7TEER (IEC-18) vs. RC IC50 0.80 1.6TEER (IEC-18) vs. RC LD50 0.86 7.6

    R2=coefficient of determination of regression analysis; m=slope of the line.ANOVA analyses were significant (P

  • emission wavelengths for LY are 430 nm and 540 nm, and forFITC-dextran, 487 nm and 518 nm, respectively. Relative cellpermeability was expressed as a percent of untreated controlgroups.

    2.6. Statistical analysis

    Chemicals used in these studies were suggested by theRegistry of Cytotoxicity (RC, Halle, 2003) and are based on theverification of the data set (RC-II), and for their validity inestablishing a correlation model between LD50's and IC50's(ICCVAM publication 01-4500, 2001a,b). IC50's were extrap-olated from concentrationeffect curves using linear regressionanalysis. The coefficient of determination (R2), slope (m), inter-cept (b) and t-statistic (two-tailed paired Student's t-test withthe more stringent equal variances assumption) were calculated.All experiments were repeated at least three times. The values infigures are expressed as percentage of untreated controls.

    In addition, the regression calculated for the AC/PP modelwas compared to the RC regression (ICCVAM GuidanceDocument, NIH publication No. 01-4500, 2001a,b) to compareour model cytotoxicity test to the RC prediction model. The RCregression equation is based on the following formula:

    logLD50 0:435 logIC50 0:625

    regression line obtained with our model parallels the RCregression and is within log 5 interval, then the test is consid-ered suitable to generate IC50 data for estimating starting doses.

    3. Results

    3.1. Comparison of cytotoxicity data and TEER measurements

    Table 1 compares IC50's, generated from AC and TEERstudies using IEC-18 cells, with those previously obtained fromCaco-2 cells. Cytotoxicity was determined using MTT cellviability assay on confluent IEC-18 and Caco-2 cells in 96-wellplates; TEER was measured in confluent monolayers grown onfilter membranes, as described above. IC50's were calculatedfrom regression analyses (not shown). Table 3 summarizes thestatistical analyses of the data in Table 1. The coefficient ofdetermination (R2) and the slope (m) of the lines of best fit areindicated. R2 measures the degree of correlation between thesets of data, while the slope is an indication of the deviation ofthe plot of experimental values from 1:1 (mM/mM) relation-ship. ANOVA calculations revealed that significant differencesexist among the data sets; further statistical comparisonsshowed no significant differences between groups (two-tailedpaired Student's t-test, P>0.05, Tables 1 and 3).

    Upon closer inspection, the regressions indicate that there ishigh correlation between the MTTand TEER values (R2 =0.99).

    179R. Konsoula, F.A. Barile / Journal of Pharmacological and Toxicological Methods 55 (2007) 176183where R2 =0.67 for 347 chemicals in the RC database(Spielmann, Genschow, Leibsch, & Halle, 1999). If theFig. 2. MTT and TEER IC50's in IEC-18 vs. RC database IC50's. Graphs of: (A) 24-hLD50's, (C) 24-h TEER IC50's for IEC-18 vs. RC IC50's, and (D) 24-h TEER IC50'In addition, the slope (m=0.23) suggests that the MTT assay ismore sensitive than TEER measurements (in the calculation ofMTT IC50's for IEC-18 vs. RC IC50's, (B) 24-h MTT IC50's for IEC-18 vs. RCs for IEC-18 vs. RC LD50's.

  • slope on log scale, when m is less than 1.0, the line is shifted tothe right, making the y-values less than x-values). This suggeststhat cell viability is affected before membrane integrity iscompromised. Good correlations were also obtained betweenIEC-18 and Caco-2 cells line. (R2 =0.75 and R2 =0.73, Table 3).Both cell lines showed the same sensitivity towards 24-h MTTand TEER (m=1.06 and m=0.99, respectively). Our AC/TEERanalysis thus allows for the differentiation between theconcentrations necessary for AC and those needed to interferewith resistance. These results suggest that the MTT assay is amore sensitive indicator of chemical exposure than TEERmeasurements. In fact, the model thus far reveals that at equalIC50's, mitochondrial activity is more likely to be altered beforeparacellular permeability is compromised.

    3.2. Comparison of cytotoxicity data and RC human data

    Table 2 summarizes the IC50's for MTT assay for IEC-18cells and the LD50's and IC50's from the RC database (registryof cytotoxicity). Statistical analysis of the data is presented inTable 3. There is a good correlation between MTT for IEC-18and LD50's (R

    2 =0.82) and IC50's (R2 =0.77) from RC,

    respectively. This suggests that IC50's from our data areconsistent with the RC database. The slope of line (m=0.36)indicates that IC50's from the MTT assay and IEC-18 cells ismore sensitive than IC50's from the RC database. Moreover, as

    there is good correlation (R2 =0.80 and 0.86, respectively). (Itshould be noted that the IC50's from the RC database use theneutral red uptake assay as a cell viability marker.) There is nostatistical difference between the data values using Student's t-test (P

  • moderate transport of markers at IC50's determined for theviability assays; paracellular transport of all markers closelyparallels the fall in TEER.

    4. Discussion

    In the present study, we investigated the suitability of the ratileac finite cell line, IEC-18, as in vitro model to differentiatebetween AC and PP. Previously, we reported that the Caco-2 cellline is useful as an in vitro model to distinguish betweenconcentrations needed for AC and PP (Konsoula & Barile, 2005).In fact, we noted that after testing 20 chemicals, cell viabilitydecreases before membrane integrity is compromised. Otherinvestigators have also shown that Caco-2 cells grown on insertsare fully differentiated and represent a good model to studyepithelial permeability and the integrity of the cell barrier(Hidalgo, Raub, & Borchardt, 1989). Caco-2 cells, however,originate from immortal colon cells, thus displaying a hightransepithelial electrical resistance reminiscent of colon epitheli-um, as well as sufficient defense against toxic insult. Conse-quently, we decided to investigate a normal rodent intestinal cellline for its the ability to establish an epithelial monolayer withtight junction properties similar to that of non-neoplastic intestine,and that can display sensitivity to respond to toxic exposure.

    The results obtained from IEC-18 cells grown on cell cultureinserts reveal that they form intestinal epithelial membranes with

    electrical resistance comparable to that of the small bowel. Themodel also demonstrates that there is direct correlation betweenthe disruption of the epithelial barrier and PP. Increased tran-sepithelial transport of markers resulted from a disruption ofmembrane integrity. Also, as with the Caco-2 cells, IEC-18 cellsare as sensitive to toxic insult as immortal colon cells, and cellviability decreases before membrane integrity is compromised.Moreover, both cell lines reveal similar TEER responses. In-terestingly, IEC-18 cells are uniformly permeable to all markersin contrast to Caco-2, the latter having displayed more favorablepassage of [3H]-D-mannitol.

    Differences in absolute TEER values registered for IEC-18and Caco-2 clearly account for the variation in marker transport.For instance, Duizer et al. (2002) and Noteborn, Steensma, andKuiper (2004) similarly reported that paracellular transport inIEC-18 cells is higher compared to Caco-2 cells. The highertransport in IEC-18 cells may be explained by the lowerresistance of epithelial barriers of the small intestine (He et al.,1998). The lower, or lack of, expression of junctional proteins(ZO-1, occludin, e-cadherin) by IEC-18 suggests that junctionalcomplexes are less tightly organized than in Caco-2 (Quaroni etal., 1979).

    We obtained high regression values between our IC50's andTEERmeasurements (Table 3), indicating that PP varies directlyand inversely with AC. According to the regression standards forthe RC database, cytotoxicity is predicted by the IEC model. In

    dih

    181R. Konsoula, F.A. Barile / Journal of Pharmacological and Toxicological Methods 55 (2007) 176183Fig. 4. Effect of (A) cadmium chloride, (B) cupric sulfate, (C) sodium dichromate

    to IC50's generated from MTT and TEER experiments. Scale for % of control for PPright axis. All control values are set at 100%. FITC=fluorescein isothiocyanate-deresistance.ydrate, and (D) ibuprofen, on PP in IEC-18 cell line. Concentrations correspond

    markers (bars) are on left axis; for TEER measurements (solid line), scale is onxtran, LY=lucifer yellow, 3H=[3H]-mannitol, TEER=transepithelial electrical

  • addition, the slopes for the MTT assay (0.23 and 0.36, Table 3)vs. TEER measurements and RC IC50's are less than 1.0,suggesting that this cell viability assay is more sensitive. Othercomparisons indicate that the assays are either comparable(m=1.06 or 0.99), or are less sensitive than the values derivedfrom the RC database (1.7, 1.6, and 7.6, Table 3). Consequently,determination of R2 is not sufficient for determining sensitivityof in vitro systems. Since paracellular transport of tight junctionmarkers correlates precisely with decreases in TEER, the datasuggests that interference with cell viability is more likely tooccur before PP is compromised. (It is important to note thatalthough some chemicals may selectively interfere with theparacellular route, overall, the experiments are not designed toaddress the mechanisms of acute toxicity. Nor will the protocoldifferentiate between passive transcellular and paracellulartransport. The study is constructed, however, to determine theassociation between PP and cell viability. As a result, calculationof a standardized formula from a larger set of compounds woulddilute any specific mechanism of toxicity.) Thus, our in vitromodel allows for the differentiation between the concentrationsnecessary for AC and those needed to interfere with PP.

    Our study represents a systematic approach to correlating atraditional pharmacokinetic system with in vitro cytotoxicitytesting procedures. Based on these results, we recommend thatthe model using IEC cells, together with the Caco-2 cell line, beused as part of a battery of in vitro tests to effectively screen forin vivo toxicity. In fact, we have computed a formula for

    predicting starting doses to estimate LD50's in toxicity testing,based on the calculations of the averages of the two mostsensitive slopes (m=0.23 and 0.36, Table 3) and correspondingy-intercepts (not shown):

    logmmol=kg LD50 0:29logmM IC50 5:9

    This standardized approach can be used in addition topreviously proposed calculations (Konsoula & Barile, 2005), toimprove the predictive ability for acute oral in vivo toxicity.Interestingly, knowledge of a compound's intestinal permeabil-ity or absorption, may not necessarily be useful to furtherclassify cytotoxicity. Comparison of our data with availablepermeability indexes for some of the compounds tested (Ingels,Beck, Oth, & Augustijns, 2004; Ruell, Tsinman, & Avdeef,2003) revealed no correlation (data not shown), suggesting thatpermeability indexes and cytotoxicity are not necessarilyrelated. Also, variability in expression of transporter/effluxsystems has been shown to alter intracellular concentrations oftoxic agents, and should be taken into account in the validationof specific classes of compounds (Behrens & Kissel, 2003).

    Our models thus incorporate and compare at least 2 exposuretimes, 3 assays, and 2 cell lines, the combination of which asingle method is not relied upon to effectively identify a toxicsubstance. Consequently the case has been repeatedly advancedthat a selective battery of validated (and normalized) tests wouldbe to the advantage of any in vitro toxicology program.

    182 R. Konsoula, F.A. Barile / Journal of Pharmacological and Toxicological Methods 55 (2007) 176183Fig. 5. Effect of: (A) nickel chloride, (B) quinine HCl, (C) trichlorfon and (D) lithiumfrom MTT and TEER experiments. Scale for % of control for PP markers (bars) are ovalues are set at 100%. FITC=fluorescein isothiocyanate-dextran, LY=lucifer yellosulfate, on PP in IEC-18 cell line. Concentrations correspond to IC50's gatheredn left axis; for TEER measurements (solid line), scale is on right axis. All controlw, 3H=[3H]-mannitol, TEER=transepithelial electrical resistance.

  • Acknowledgements

    This work was supported in part by grants from the NIH,NIEHS (R15 ES012170-01), USA.

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    Correlation of in vitro cytotoxicity with paracellular permeability in mortal rat intestinal c.....IntroductionMaterials and methodsMaterialsCell cultureMTT cell viability assayTransepithelial electrical resistance (TEER)Paracellular permeability (PP) of mannitol, lucifer yellow and fluorescein-dextranStatistical analysis

    ResultsComparison of cytotoxicity data and TEER measurementsComparison of cytotoxicity data and RC human dataComparison of TEER and paracellular permeability (PP)

    DiscussionAcknowledgementsReferences

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