Correlation of in vitro cytotoxicity with paracellular permeability in Caco-2 cells
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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: firstname.lastname@example.org (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
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
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
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
; 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
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)
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
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-
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
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 b...