DETECTION OF MXR-INHIBITING PROPERTIES OF CHEMICALS AND
ENVIRONMENTAL SAMPLES – “Pgp- INHIBITION ASSAY”
I.
Principle
The idea is to measure the P-glycoprotein-inhibiting potential of different chemicals
and environmental samples. As an in vitro bioassay tool a specific cell line is used: NIH 3T3
mouse fibroblasts stable transfected with human MDR1 gene (NIH 3T3/MDR1), in pair with
non-transfected (NIH3T3) cells.
The principle of the method is that Pgp-inhibitors can modulate calcein accumulation
in NIH 3T3/MDR1 transfected cells. Calcein acetoxymethyl ester (CaAM) is a substrate for
Pgp, and as a highly lipid soluble dye rapidly penetrates the cell membrane, practically is
non-fluorescent, but intracellular esterase’s transform the dye to a hydrophilic and intensively
fluorescent acid form, calcein, whose fluorescence is essentially insensitive to changes in
pH, as well as to the changes in Ca2+ or Mg2+ concentration. Calcein is well retained by the
cells, with no apparent cytotoxicity, and is not exported by the Pgp. These properties of
CaAM dye in combination with MDR1 transfected cells are developed and standardized as a
Pgp-inhibition assay. The assay is performed on 96 well plates.
Cell Lines.
NIH 3T3 mouse fibroblasts and human MDR1-stable transfected NIH 3T3 fibroblasts were kindly
provided by Dr. B. Sarkadi from the National Institute of Hematology, Blood Transfusion and Immunology,
Budapest, Hungary with permission of Dr. M. M. Gottesman from Laboratory of Cell Biology, National Cancer
Institute of Health, Bethesda, Maryland, U.S.A. The expression of transmembrane ABC P-glycoprotein and
characterization of the multidrug resistance phenotype of NIH 3T3/MDR1 cells have been described previously
(Bruggermann et al., 1992.). Cell culturing was performed under standard conditions in DMEM (Sigma, St. Louis,
USA) supplemented with 10% fetal calf serum (Gibco BRL, Paisley, UK) in a humidified atmosphere consisting of
95% air and 5% CO2 at 37°C.
II.
1.
2.
3.
4.
5.
6.
Procedure
Seed NIH 3T3 and NIH 3T3/MDR1 cells at the density of 6 x 104 cells per well in a
96 well plate and allow to attach overnight. In the morning, remove the
medium
and wash cells with 1 x 200 l of HPMI warm medium (37 C)
Add 100 l of HPMI medium to the cells
From stock solutions of chemicals and environmental extracts prepare serial
dilutions in HPMI
Add 50 l of serial dilutions in triplicate to the cells on a plate (avoid merges) and
preincubate for 15 – 20 min on 37 C
Prepare warm CaAM solution, e.g. 5 ml HPMI + 50 l CaAM (100 M stock in
DMSO from -20°C)
After preincubation add 50 l of CaAM solution to the plates and incubate on 37 C
Measurement
7.
Every 15 min measure fluorescence of accumulated calcein by using excitation 485
and emission 530 filters. Between two measurements keep plates in the
incubator on 37 C in the dark. If concentration of calcein in the cells treated
with particular sample is not rising after 45 min, add high concentration of
verapamil (100 M) to check if this is a false negative result. If fluorescence units
do not rise after verapamil is added probably there are esterase inhibitors in
tested sample. In that case another fluorophore, Rhodamine 123 should be
used, with minor revisions of the protocol (final conc. 1 M, incubation time 2 h).
8.
After 45 min (the last measurement), decant the medium from the plate and
add
100 l of 0.1 % Triton X-100 to each well. After 10 min measure the fluorescence and
take these values as the final end point data.
Subtract control (solvent) from all end point data and plot the data for verapamil.
To determine the Pgp-inhibiting potency of a particular environmental sample
(extract) the fluorescent response has to be interpolated in a dose-response curve
for the reference chemical – verapamil (VER). For a reliable determination the
response has to fit on the linear part of the VER dose-response curve. The Pgpinhibitory potency of either a compound or environmental extract is then expressed
in m VER-equivalents.
9.
III.
Chemicals
HPMI medium
pH 7,4
120 mM NaCl
for 40 ml
58,44 mg /ml 1M
7 mg / ml 120 mM
198,17 mg/ml 1M
1,98 mg/ml 10mM
141,97 mg/ml 1M
0,71 mg/ml 5mM
280,0 mg
10 mM HEPES
pH 7,4
260,3 mg/ml 1M
2,6 mg/ml 10 mM
104,0 mg
5 mM KCL
74,56 mg/ml 1M
0,37 mg/ml 5mM
14,8 mg
0,4 mM MgCL2
228 mg/ml 1M
91 g/ml 0,4 mM
40 mM
91 mg/10 ml
40 l
0,04 mM CaCL2
110,99 mg/ml 1M
4,4 g/ml 0,04
mM
84,01 mg/ml 1M
840 g/ml 10 mM
40 mM
44 mg/10 ml
40 l
10 mM D-glucose
5 mM Na2HPO4
10 mM NaHCO3
IV.
Stock
solutions
79,2 mg
28,4 mg
33,6 mg
References
1. Hollo Z., Homolya L., Wiliam-Davis C. and Sarkadi B., Calcein accumulation as a fluorometric
functional assay of the multidrug transporter, Biochem. Biophys. Acta, 1494, 384, 1994.
2. Smital T., Pivčević B. and Kurelec B., Reversal of multidrug resistance by extract from the
3.
4.
marine alga Caulerpa taxifolia, Period. Biol., 98, 197, 1996.
Yoshimura A., Shudo N., Ikeda S., Ichikawa M., Sumizawa T and Akiyama S.I., Novel
screening method for agents that overcome classical multidrug resistance in a human cell line,
Cancer. Lett., 45, 50, 1990.
Pivčević, B., Kurelec, B., and Müller, W.E.G., Measurement of water pollutants with
multixenobiotic resistance inhibiting properties, Use of aquatic invertebrates as tools for
5.
6.
monitoring of environmental hazards, Műller W. E. G., Gustav Fischer Verlag, Stuttgart, 1994,
129.
Kurelec, B., Pivčević, B., Műller, W. E. G., Determination of pollutants with multixenobioticresistance inhibiting properties, Mar. Environ. Res., 39, 261, 1995.
Kurelec, B., Smital, T., Pivčević, B., Eufemia, N., and Epel, D., Multixenobiotic resistance, Pglycoprotein, and chemosensitizers, Ecotoxicology, 9, 307-327, 2000.