Photophysical characterization and flow cytometry applications of cholylamidofluorescein, a fluorescent bile acid scaffold.
ABSTRACT Cholylamidofluorescein (CamF) has been selected as a fluorescent bile acid scaffold to perform a full characterization of its photophysical properties. In aqueous medium, under nitrogen, the absorption spectrum of CamF was expectedly dependent on pH. Under air, the presence of CO(2) resulted in a partial protonation of the photoactive form, reducing the absorbance of CamF. The fluorescence spectrum of CamF in ethanol (lambda(exc) = 481 nm) showed a broad band with maximum at 518 nm; the fluorescence quantum yield was 0.67, and the fluorescence lifetime was 4.8 ns. Laser flash photolysis of CamF showed the triplet state transient with a broad maximum at ca. 540 nm and a lifetime of 19 mus. Flow cytometric kinetic assay of CamF uptake in real time was performed in suspensions of rat hepatocytes, showing that living hepatocytes accumulated slowly but constantly CamF along the 5-minute experimental period. Besides, intracellular fluorescence of live cells was found to be clearly dependent on the extracellular concentration of CamF. Thus, flow cytometry has allowed us to demonstrate that CamF is specifically taken up by living rat hepatocytes in a concentration-dependent fashion, suggesting the suitability of this molecule for further studies on bile-acid transport in liver cells.
Article: Drug-induced cholestasis.[show abstract] [hide abstract]
ABSTRACT: Drug-induced cholestasis may be due to impairment of hepatocellular bile secretion (pure cholestasis or cholestatic hepatitis), obstruction of ductules (cholangiolitis) or interlobular ducts (cholangitis), or extrahepatic obstruction (sclerosing cholangitis). Mechanisms of hepatocellular cholestasis are multiple and include inhibition of various transport systems, cytoskeleton poisoning, disturbed intracellular calcium homeostasis and increased permeability with regurgitation of bile constituents into plasma. Pure hepatocellular cholestasis is mostly observed with sex steroid hormones and anabolic steroids. Ductular or ductal cholestasis (drug-induced cholangiopathy) may be acute and self-limited, or prolonged with ductopenia, occasionally leading to biliary cirrhosis. An immune mechanism has been proposed. Sclerosing cholangitis with strictures near the confluent of hepatic ducts is observed after intraarterial administration of floxuridine for chemotherapy of hepatic metastases. Some drugs may induce the formation of cholesterol gallstones, or precipitate in bile and form biliary sludge or stones in the gallbladder or common bile duct.Journal of Hepatology 02/1997; 26 Suppl 1:1-4. · 9.86 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The macrocyclic “cholaphanes” 3a−c were synthesized from the inexpensive steroid cholic acid. Like earlier relatives they feature substantial cavities with inward-directed hydroxyl groups, suitable for binding polar molecules such as carbohydrates in nonpolar media. New features are the externally directed alkyl chains, promoting solubility in organic solvents, and (in the case of 3b/c) reduced conformational freedom resulting from truncation of the steroidal side-chain. In particular, modeling shows that the smallest macrocycle 3c possesses very little flexibility, preferring an open conformation which is also revealed in the X-ray crystal structure of its pentahydrate. NMR studies indicated that all three cholaphanes form 1:1 complexes with octyl β-d-glucoside in CDCl3, with Ka = 600−1560 M-1. Cholaphanes 3b/c proved able to extract methyl β-d-glucoside from aqueous solutions into CHCl3. The transport of methyl β-d-glucoside across a chloroform barrier was also demonstrated for 3c.The Journal of Organic Chemistry 11/1997; 62(24). · 4.56 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: To better characterize the function of the ABCG2 transporter in vitro, we generated three cell lines (MXRA, MXRG, and MXRT) stably expressing ABCG2 after transfection of wild-type ABCG2 and two mutants (R482G and R482T), respectively. ABCG2 expression and function were analyzed by flow cytometry using monoclonal antibodies, a variety of fluorescent substrates, and a series of potential inhibitors of the transporter. ABCG2 expression was detected in all cell lines. The cell lines effluxed mitoxantrone (MXR), but only the mutants effluxed rhodamine 123 (Rho123), SYTO13, doxorubicin, and daunorubicin. After incubation with MXR, intracellular accumulations were 9- and 22-fold higher in MXRA than in MXRT and MXRG cells, respectively, suggesting that ABCG2 also modulates the influx rate of the drug. Flow cytometry kinetic studies of MXR efflux showed that MXRG cells effluxed 50% of the drug at a faster rate than MXRA and MXRT cells (t50: 15.3 min vs. 27.8 and 44.5 min, respectively). MXRG cells also extruded Rho123 and SYTO13 at a faster rate than MXRT cells. ABCG2-mediated transport was inhibited by fumitremorgin C, cyclosporine A, and PSC-833, but not by verapamil or probenecid. MXRG cells displayed the highest level of resistance to MXR, doxorubicin, and daunorubicin in the cytotoxicity assays. Glycine mutations at position 482 have a significant impact on ABCG2 function by modifying its substrate specificity and its influx/efflux rates. This study also demonstrates that flow cytometry constitutes a powerful tool for the kinetic analysis of ABC transporters.Cytometry Part A 01/2005; 62(2):129-38. · 3.71 Impact Factor
PAPER www.rsc.org/pps | Photochemical & Photobiological Sciences
Photophysical characterization and flow cytometry applications of
cholylamidofluorescein, a fluorescent bile acid scaffold
Jana Rohacova,aM. Luisa Marin,aAlicia Mart´ ınez-Romero,bJos´ e-Enrique O’Connor,b
M. Jose Gomez-Lechon,c,dM. Teresa Donato,c,d,eJose V. Castellc,d,eand Miguel A. Miranda*a
Received 16th April 2008, Accepted 29th May 2008
First published as an Advance Article on the web 12th June 2008
Cholylamidofluorescein (CamF) has been selected as a fluorescent bile acid scaffold to perform a full
characterization of its photophysical properties. In aqueous medium, under nitrogen, the absorption
spectrum of CamF was expectedly dependent on pH. Under air, the presence of CO2resulted in a
partial protonation of the photoactive form, reducing the absorbance of CamF. The fluorescence
spectrum of CamF in ethanol (kexc= 481 nm) showed a broad band with maximum at 518 nm; the
fluorescence quantum yield was 0.67, and the fluorescence lifetime was 4.8 ns. Laser flash photolysis of
CamF showed the triplet state transient with a broad maximum at ca. 540 nm and a lifetime of 19 ls.
Flow cytometric kinetic assay of CamF uptake in real time was performed in suspensions of rat
hepatocytes, showing that living hepatocytes accumulated slowly but constantly CamF along the
5-minute experimental period. Besides, intracellular fluorescence of live cells was found to be clearly
dependent on the extracellular concentration of CamF. Thus, flow cytometry has allowed us to
demonstrate that CamF is specifically taken up by living rat hepatocytes in a concentration-dependent
fashion, suggesting the suitability of this molecule for further studies on bile-acid transport in liver cells.
Efficient development of new pharmaceuticals requires the avail-
ability of human relevant toxicology information early in the
selection process. In this sense, long-term hepatotoxicity is es-
pecially difficult to predict and relies almost exclusively on animal
experimentation. However, the use of animal models is expensive,
time consuming, raises ethical issues and may not be extrapolated
Cholestasis is a particular type of hepatotoxicity, consisting in
the failure of bile to reach the duodenum.3,4This is connected with
bile acids transport, which involves their uptake from the blood
and subsequent excretion to the bile canaliculi.5
Numerous ex vivo or in vitro models are available for exploring
the function and regulation of hepatic transporters, including
perfused liver,6liver slices,7,8primary hepatocyte cultures (either
as conventional monolayers with no overlay or as sandwich
cultures),9primary hepatocyte suspensions10and transfected cell
lines expressing defined transporters.11
The identification of which transporters are involved in the
uptake and efflux of a particular molecule requires a model that
maintains physiological expression of most, if not all of them. In
aInstituto de Tecnolog´ ıa Qu´ ımica-Departamento de Qu´ ımica (UPV-CSIC),
Avda de los Naranjos s/n, E-46022, Valencia, Spain. E-mail: mmiranda@
qim.upv.es; Fax: +34963877809; Tel: +34963877807
bLaboratorio de Cit´ omica, Unidad Mixta CIPF-UVEG, Centro de Investi-
gaci´ on Pr´ ıncipe Felipe, Valencia, Spain
cUnidad de Hepatolog´ ıa Experimental, Centro de Investigaci´ on Hospital
Universitario “La Fe”, Valencia, Spain
dCIBERHEPAD, FIS, Spain
eDepartamento de Bioqu´ ımica y Biolog´ ıa Molecular, Facultad de Medicina,
Universidad de Valencia, Spain
this context, freshly isolated or short-term cultures (i.e. 2 h after
plating) have proven useful for uptake studies.12,13
As regards the most commonly employed methods to evaluate
drug effects on the uptake of bile acids, they are based on
radiolabeled compounds14–17or on fluorescent derivatives of
bile acids.18–24Among the latter, cholic, chenodeoxycholic or
ursocholic acids have been conjugated (either directly or through
an amino acid spacer) with fluorescein, nitrobenzoxadiazole or
other fluorophores. The standard analytical methodology relies
on emission measurements, thus making use of the singlet excited
state properties of the selected derivatives.
However, the possibilities of excited states as reporters for
analytical purposes are not limited to fluorescence detection
or quantitation. In fact, lifetime determinations in cellular sys-
tems have been successfully applied to time-resolved fluores-
cence microscopy.25–30Moreover, triplet excited states can also
be investigated in complex biological media, including cells;
their longer lifetimes provide a wider dynamic range, which
could be potentially exploited for the development of new
analytical tools.31Hence, it makes sense to perform a full
photophysical characterization of the fluorescent derivatives of
bile acids employed for uptake studies. Such a characterization
should include fluorescence quantum yields, singlet energies and
lifetimes as well as triplet transient absorption spectra and
With this background we have chosen a fluorescent bile
acid scaffold, N-fluoresceinyl-3a,7a,12a-trihydroxy-5b-cholan-24-
amide (cholylamidofluorescein or CamF) to perform a full char-
acterization of its photophysical properties. In addition, we have
used CamF in a flow cytometry kinetic assay based on the uptake
of the fluorescent compound in fresh suspensions of hepatocytes.
860 | Photochem. Photobiol. Sci., 2008, 7, 860–866This journal is © The Royal Society of Chemistry and Owner Societies 2008
appropriate conditions is taken up and retained by isolated rat
hepatocytes in suspension.
Materials and methods
Cholic acid, aminofluorescein (amF), collagenase, DMSO, fluo-
rescein and propidium iodide were provided by Sigma Chemical
Co. (Madrid, Spain). Ethanol (99.9%) was from Merck (Darm-
stadt, Germany). Ham’s F-12 and Lebovitz L-15 medium and
calf serum were acquired from Gibco (Madrid, Spain). All other
chemicals used for the synthesis of the fluorescent derivatives were
reagent grade from Sigma-Aldrich and were used as received.
Nuclear magnetic resonance
(Rheinstetten, Germany) 300 MHz instrument. Frequencies are
reported in hertz, CDCl3and CD3OD were used as solvents, and
the signal corresponding to the deuterated solvent, in each case,
was taken as the reference: CDCl3(d = 7.26 for
77.2 for13C NMR) and CD3OD (d = 3.31 for1H NMR, d = 49.0
1H NMR, d =
V-530 spectrometer (Japan). To measure absorption dependence
of fluorescein derivatives versus pH, solutions at a concentration
Fluorescence spectra were recorded on a FS900 fluorimeter,
and lifetimes were measured with a FL900 setup, both from
Edinburgh Instruments (Reading, UK). Lifetime measurements
(1.5 ns pulse width) as excitation source. The kinetic traces were
fitted by monoexponential decay functions using a re-convolution
procedure to separate signals from the lamp pulse profile. The
solutions were purged with nitrogen for at least 15 min before
the measurements. When influence of oxygen or carbon dioxide
was examined solutions were purged with these gases for 15 min
prior to the measurement. The absorbance of the solutions at the
path length were employed, and experiments were performed at
Laser flash photolysis studies in the kinetic mode were carried
out with a pulsed Nd:YAG SL404G-10 Spectron Laser Systems
(Spectron Laser System; Rugby England) at the excitation wave-
length of 355 nm. The single pulses were ∼10 ns duration, and the
energy was lower than 10 mJ pulse−1. The detecting light source
was a pulsed Lo255 Oriel xenon lamp. The laser flash photolysis
system consisted of the pulsed laser, the Xe lamp, a 77200 Oriel
monochromator, an Oriel photomultiplier tube (PMT) system
made up of a 77348 side-on PMT tube, 70680 PMT housing and
a 70705 PMT power supply (Oriel Corporation, Stratford CT).
The oscilloscope was a TDS-640A Tektronix (Tektronix-Holland
N.V., Heerenven, The Netherlands). The output signal from the
oscilloscope was transferred to a personal computer. In a typical
experiment a solution of CamF or N-fluoresceinylacetamide
(AcF) in ethanol was placed in a sealed cuvette at an appropriate
concentration to give an absorbance of ca. 0.4 at the excitation
wavelength (kexc= 355 nm). All measurements were performed at
room temperature in 1 cm optical path length cuvettes.
Synthesis of 3a,7a,12a-triformyloxy-5b-cholan-24-oic acid.
was prepared from cholic acid as described in the literature.32
Briefly, to a solution of cholic acid (1.00 g, 2.45 mmol) in formic
acid 88% (4 ml, 103 mmol) 0.5 ml of perchloric acid were added,
and the solution was heated at 50◦C for 2 h. Then 3.2 ml of acetic
anhydride were slowly added, and the mixture was heated at 60◦C
was cooled to room temperature, poured into 40 ml of cold water,
and the resulting precipitate was collected by filtration as a white
solid. The crude product was recrystallized from ethanol–water.
The white crystalline solid was filtered and dried to yield 1.05 g
(87%) of 3a,7a,12a-triformyloxy-5b-cholan-24-oic acid.1H NMR
(CDCl3): d 8.16 (s, 1H, 12a-OOCH), 8.10 (s, 1H, 7a-OOCH), 8.02
(s, 1H, 3a-OOCH), 5.27 (br s, 1H, H-12b), 5.07 (br s, 1H, H-7b),
4.71 (m, 1H, H-3b), 0.94 (s, 3H, 19-CH3), 0.85 (d, J = 6.6 Hz,
3H, 21-CH3), 0.76 (s, 3H, 18-CH3).13C NMR (CDCl3): d 179.6
(CH), 73.8 (CH), 70.7 (CH), 47.2 (CH), 45.0 (C), 43.0 (CH), 40.8
(CH2), 30.9 (CH2), 30.4 (CH2), 28.5 (CH), 27.1 (CH2), 26.6 (CH2),
25.5 (CH2), 22.7 (CH2), 22.3 (CH3), 17.4 (CH3), 12.1 (CH3).
Synthesis of N-fluoresceinyl-3a,7a,12a-trihydroxy-5b-cholan-
24-amide (cholylamidofluorescein or CamF).
(86 ll, 1 mmol, 2 eq) was added dropwise to a solution of
3a,7a,12a-triformyloxy-5b-cholan-24-oic acid (0.25 g, 0.5 mmol)
in 20 ml of anhydrous benzene under nitrogen. The resulting
solution was stirred at room temperature for 4 h, and the solvent
was evaporated giving a pale yellow oil. The crude product was
a solution of fluoresceinamine (0.09 g, 0.25 mmol, 0.5 eq) in
7 ml of anhydrous acetone was slowly added. The resulting
mixture was stirred overnight at room temperature in absence
of light. Then solvent was removed, and the crude containing
used in the following step without any further purification.
The crude product was dissolved in 10 ml of 1 M KOH–MeOH
and stirred at room temperature for 2 h. The resulting red solution
was poured into 40 ml of 1 M HCl, and the obtained precipitate
was filtered, washed with water and dried. The crude was purified
by column chromatography on silica gel using methanol–ethyl
orange solid.1H RMN (CD3OD): d 8.32 (s, 1H, 5?-CH(Ar?)), 7.85
(d, J = 8.2 Hz, 1H, 3?-CH(Ar?)), 7.14 (d, J = 8.2 Hz, 1H, 2?-
CH(Ar?)), 6.52–6.67 (m, 6H, Ar), 3.98 (m, 1H, H-12b), 3.80 (m,
1H, H-7b), 3.38 (m, 1H, H-3b), 1.10 (d, J = 6 Hz, 3H, 21-CH3),
0.92 (s, 3H, 19-CH3), 0.74 (s, 3H, 18-CH3).13C RMN (CD3OD): d
175.7 (C), 171.4 (C), 154.3 (C), 142.0 (C), 130.3 (CH), 128.0 (CH),
125.9 (CH), 116.2 (CH), 113.8 (CH), 111.7 (C), 103.6 (CH), 74.1
(CH), 40.5 (CH2), 37.0 (CH), 36.6 (CH2), 36.0 (CH2), 35.0 (CH2),
33.1 (CH2), 31.0 (CH2), 29.7 (CH2), 28.8 (CH2), 28.0 (CH), 24.3
(CH2), 23.2 (CH3), 17.9 (CH3), 13.1 (CH3).
This journal is © The Royal Society of Chemistry and Owner Societies 2008Photochem. Photobiol. Sci., 2008, 7, 860–866 | 861
To a solution of fluoresceinamine (50 mg, 0.14 mmol) in
and the mixture was refluxed for 1 h. After removal of the solvent,
the crude product was purified by column chromatography on
silica gel using methanol–ethyl acetate (15:85) as eluent, to yield
the acetamide (46 mg, 82%) as a yellow crystalline solid (melting
point 240◦C with decomposition) (lit. 237–240◦C).33 1H RMN
(CD3OD): d 8.30 (s, 1H, 5?-CH (Ar?)), 7.83 (d, J = 8.3 Hz, 1H,
3?-CH (Ar?)), 7.14 (d, J = 8.3 Hz, 1H, 2?-CH (Ar?)), 6.52–6.67
(m, 6H, Ar), 2.19 (s, 3H, CH3CO-).13C RMN (CD3OD): d 172.0
(C), 171.3 (C), 154.2 (C), 141.9 (C), 130.2 (CH), 128.0 (CH), 125.8
(CH), 116.1 (CH), 113.7 (CH), 111.6 (C), 103.5 (CH), 24.2 (CH3).
Rat hepatocyte isolation
Hepatocytes were obtained from 200–300 g Sprague Dawley
male rats by perfusion of the liver with collagenase as described
elsewhere.34Cell viability of suspension, assessed by the trypan
blue exclusion test, was higher than 85%.
Flow cytometry of CamF transport
Suspensions of freshly isolated rat hepatocytes were diluted at
a density of 105viable cells ml−1in Ham’s F-12/Lebovitz L-
15 (1:1) medium supplemented with 2% calf serum, 10 nM
insulin plus 0.2% bovine serum albumin and kept at 37◦C in
a 5% CO2humidified atmosphere until analysis. Flow cytometric
experiments were always performed within 2 h of cell isolation.
For flow cytometric analyses CamF was dissolved in ethanol at
1.7 mM (1 mg ml−1). In the kinetic experiments of CamF uptake,
hepatocyte suspensions (105viable cells ml−1) were dispersed in
standard polypropylene tubes and stained with propidium iodide
to identify dead cells.35Then, each tube was loaded in the flow
cytometer and acquisition was started for about 10 s, in order to
detect the green autofluorescence of cells at the initial time. Then,
the data acquisition was paused and an appropriate volume of
stock CamF solution added quickly to a final concentration of
100 nM. At this point data acquisition was continued until 300 s.
Transport of CamF inside the cell was detected and quantified by
measuring the increase of green fluorescence in cells along time.
Single end-point flow cytometric measurements of cellular
fluorescence were performed to investigate the concentration
dependence of CamF intracellular accumulation. Freshly isolated
rat hepatocyte suspensions (105viable cells ml−1) were dispensed
in standard polypropylene tubes and incubated for 15 min with a
range of increasing concentrations of CamF for 15 min at 37◦C
in a 5% CO2humidified atmosphere and stained with propidium
iodide at 5 lg ml−1for the last 5 min to identify dead cells. Cell
suspensions were run in the flow cytometer, and fluorescence data
from 10000 live cells acquired.
In parallel, cell-free solutions of CamF in ethanol (covering
the same range of concentrations added to the cell suspensions)
Multi-Mode Microplate Reader (BioTek, Vermont, USA) using
The kinetic and the single end-point measurements were
performed in a Cytomics FC500 MCL flow cytometer (Beckman-
Coulter, Brea, CA) equipped with an air-cooled argon-ion laser
emitting at 488 nm. The fluorescence emissions of each cell were
collected at 525 nm (CamF green fluorescence) and 625 nm
angle laser light scatter (FS), an estimation of cell size, were used
for gross morphological assessment of cells and the exclusion
of debris. Data analysis in the kinetic and single end-point flow
cytometric measurements was performed using the CXP-software
(Beckman-Coulter, Brea, CA) interfaced to the flow cytometer.
Results and discussion
Conjugation of bile acids with a fluorescein unit has been previ-
ously achieved following two approaches: (i) direct reaction with
is not straightforward.
in photophysical studies, a new synthetic sequence in three steps
was designed for the preparation of CamF which ensued with a
good overall yield (49%) (see Scheme 1). It started with protection
of the three hydroxy groups as the corresponding formiates.
Then, the carboxylic acid was activated as the acyl chloride and
reacted with aminofluorescein to form the corresponding amide.
Final deprotection of the hydroxy groups afforded the desired
fluorescent cholic acid derivative.
60◦C (87%, two steps); (iii) (COCl)2, Bz, rt; (iv) amF, CH3COCH3, 0◦C
to rt; (v) KOH 1M–MeOH, rt, (56%, three steps).
In aqueous medium, under nitrogen, the absorption spectrum
of CamF was expectedly dependent on pH, as reported for other
of the two maxima at 450 and 490 nm experienced a dramatic
change within the employed pH range, with the longer wavelength
band becoming by far the predominating one above pH ≥7 (see
Fig. 1). On the other hand, the spectra in ethanolic solution
showed again a broad absorption band with two maxima at 454
and 481 nm. Under air, the presence of CO2resulted in a partial
862 | Photochem. Photobiol. Sci., 2008, 7, 860–866This journal is © The Royal Society of Chemistry and Owner Societies 2008
aqueous solutions of CamF as a function of the pH (between 2 and 12).
Inset: titration curve.
Changes of the absorption spectrum of air-equilibrated, buffered
confirmed by performing parallel experiments under O2and CO2
atmosphere (see Fig. 2).
M) under different conditions.
Absorption spectrum of an ethanolic solution of CamF (2 × 10−5
The fluorescence spectrum of CamF in ethanol (kexc= 481 nm),
under N2, showed a broad band with maximum at 518 nm. This
emission was activated by the long-wavelength (k > 400 nm)
absorption region, as revealed by the excitation spectrum, which
lacked the higher energy bands. The difference between excitation
and absorption spectra reveals that excitation at wavelength
shorter than 400 nm is less efficient in producing fluorescence
emission, probably due to other non-radiative deactivation phe-
nomena. From the intersection of the normalized excitation and
emission spectra (see Fig. 3), the energy of the singlet state (E0–0)
was estimated to be 57 kcal mol−1.
The fluorescence quantum yield of CamF in ethanol under ni-
trogen was 0.67. This was determined using the parent fluorescein
in an anaerobic solution of aqueous NaOH 0.01 M as a standard
(U = 0.85, kexc= 490 nm).38
It is well-known that amide derivatives of aminofluorescein are
highly fluorescent, whereas aminofluorescein alone has a very low
due to the intramolecular self-quenching.38Thus, the behavior of
under N2. Inset: fluorescence decay trace measured at kem= 518 nm.
Excitation and emission spectra of ethanolic solutions of CamF
CamF can be attributed to the amide nature of this compound.
This fact was confirmed by determining the fluorescent quantum
yield of acetylfluorescein (AcF) which was found to be U = 0.64
under the same experimental conditions.
Time-resolved measurements provide valuable information on
The singlet lifetime of CamF was measured by means of time
resolved fluorescence spectroscopy. The fluorescence decay trace
is shown in Fig. 3 as an inset. A good resolution was observed in
the nanosecond timescale, and the kinetic data were found to be
in excellent fitting with a monoexponential law, giving a value of
4.8 ns for the fluorescence lifetime (sS) of CamF in ethanol, under
nitrogen. As expected, a similar value was found for the closely
related N-acyl derivative AcF.
In comparison with singlet excited state properties, the pho-
tophysical behaviour of the excited triplets has been much
less exploited for analytical purposes. Typically, information on
these chemical entities can be obtained by transient absorption
spectroscopy. In this context, laser flash photolysis of CamF was
performed in ethanol under anaerobic atmosphere, using 355 nm
as the excitation source (absorbance ca. 0.4). Fig. 4 shows a
transient spectrum with a broad maximum at ca. 540 nm and
atmosphere obtained at 1 (?), 4 (●) and 10 (?) ls after laser flash
excitation (kexc= 355 nm). Inset: decay trace recorded at 540 nm.
Transient absorption spectra of CamF in ethanol under anaerobic
This journal is © The Royal Society of Chemistry and Owner Societies 2008Photochem. Photobiol. Sci., 2008, 7, 860–866 | 863
a tail extending beyond 650 nm. This transient followed a first
order decay with a lifetime of 19 ls (see inset) and was efficiently
quenched by oxygen, which supports its assignment as the triplet
is probably due to a tautomer, taking into account its slow growth
and long lifetime. Similar results were obtained upon laser flash
excitation of an ethanol solution of AcF (kmaxca. 520 nm, sT=
In addition to the positive absorption at >500 nm, negative
bands were also observed between 425 and 500 nm. These bands
can be safely attributed to depletion of the ground state species
absorbing in the same wavelength region (see Fig. 2). They
or basic pH and seems to be the only photoactive form.
In order to assess the suitability of CamF for further studies of
bile acid transport, the dynamics of CamF transport across the
orange fluorescence in rat hepatocyte suspensions stained for 10 min
with propidium iodide, as described in Materials and methods. The
biparametric dot plot shows the population of dead cells with high
R1) with low levels of propidium orange fluorescence (autofluorescence).
Cell suspensions were prepared and run in the flow cytometer as indicated
in Materials and methods. Live cells were gated in region R1 and dead
cells in region R3 of the graph in panel A. Data are the mean ± S.D. of
three independent experiments.
(A) Representative distribution of cell size (Forward Scatter) and
plasma membrane of hepatocytes was studied. For this purpose,
multiparametric flow cytometry was applied, as this cellular
methodology allows the examination in real time of multiple
fluorescences of individual cells in suspension.35In a first series
of experiments a flow cytometric kinetic assay of CamF uptake
in real time was performed in suspensions of rat hepatocytes. Cell
suspensions were stained with propidium iodide for 10 min prior
to flow cytometric analysis, to identify dead cells (Fig. 5A). In this
in a kinetic plot of green fluorescence intensity versus time. Our
results (Fig. 5B) showed that living hepatocytes accumulated
slowly but constantly CamF along the 5-minute kinetics period,
while dead cells immediately took up CamF and exhibited a much
lower and constant fluorescence, suggesting a rapid equilibration
with the fluorescent probe in the medium.
of CamF transport across plasma membrane of liver cells was
addressed. Suspensions of freshly isolated rat hepatocytes were
concentrations of CamF and stained with propidium iodide for
the analysis. In this way, only the intracellular fluorescence of live
cells was recorded (Fig. 6A) and found to be clearly dependent on
the extracellular concentration of CamF. No dose-dependence of
dence of intracellular CamF fluorescence in rat hepatocytes incubated for
15 min with the indicated CamF concentrations plus propidium iodide, as
described in Materials and methods. Bars show data only from live cells,
gated in region R1 of the graph in Fig. 5, panel A. (B) Spectrofluorimetric
determination of the concentration dependence of CamF fluorescence in
cell-free solutions of CamF in ethanol, measured with typical fluorescein
filters in a 96-well plate spectrofluorimeter, as described in Materials and
methods. Data are the mean ± S.D. of three independent experiments.
(A) Flow cytometric determination of the concentration depen-
864 | Photochem. Photobiol. Sci., 2008, 7, 860–866 This journal is © The Royal Society of Chemistry and Owner Societies 2008
fluorescence increase could be observed in dead cells (not shown).
degree of concentration-dependence of CamF fluorescence was
was measured in a plate spectrofluorimeter adjusted to the same
optical settings as the flow cytometer (Fig. 6B).
a fluorescence-based quantitative technology that allows the
examination in real time of functional properties of individual
cells.35Flow cytometric kinetic assays have been widely used to
monitor transport of fluorescent probes across cell membranes.39
This kind of experimental approach has previously been used
by some of us to investigate mitochondrial membrane potential-
driven uptake of fluorochromes40and oxidative metabolism in
isolated hepatocytes.41In this work, flow cytometry has allowed
us to demonstrate that CamF is specifically taken up by living rat
the suitability of this molecule for further studies on bile-acid
transport in liver cells.
This work was supported by the ALIVE Foundation, and funds
from the European Commission, grants LSHB-CT-2004-504761,
LSHB-CT-2004-512051 and LSSB-CT-2005-037499. Financial
support from CSIC (fellowship I3P-2005) is also gratefully ac-
Notes and references
1 A. E. M. Vickers, in In Vitro Methods in Pharmaceutical Research, ed.
J. V. Castell and M. J. Gomez-Lechon, Academic Press, London, 1997,
2 J. V. Castell, M. J. Gomez-Lechon, X. Ponsoda and R. Bort, in In Vitro
Lechon, Academic Press, London, 1997, pp. 375–410.
3 R. Poupon, O. Chazouilleres and R. E. Poupon, Chronic cholestatic
diseases, J. Hepatol., 2000, 32, 129–140.
4 S. Erlinger, Drug-induced cholestasis, J. Hepatol., 1997, 26, 1–4.
5 G. A. Kullak-Ublick, U. Beuers and G. Paumgartner, Hepatobiliary
transport, J. Hepatol., 2000, 32, 3–18.
6 M. L. Ruiz, S. S. Villanueva, M. G. Luquita, M. Vore, A. D. Mottino
and V. A. Catania, Ethynylestradiol increases expression and activity
of rat liver MRP3, Drug Metab. Dispos., 2006, 34, 1030–1034.
7 P. Olinga, I. H. Hof, M. T. Merema, M. Smit, M. H. de Jager, P. J.
Swart, M. J. H. Slooff, D. K. F. Meijer and G. M. M. Groothuis, The
hepatic drug uptake, J. Pharmacol. Toxicol. Methods, 2001, 45, 55–63.
8 M. G. L. Elferink, P. Olinga, A. L. Draaisma, M. T. Merema, K. N.
Faber, M. J. H. Slooff, D. K. F. Meijer and G. M. M. Groothuis, LPS-
induced downregulation of MRP2 and BSEP in human liver is due to a
posttranscriptional process, Am. J. Physiol., 2004, 287, G1008–G1016.
9 X. Liu, E. L. Le, Cluyse, K. R. Brouwer, L. S. Gan, J. J. Lemasters,
B. Stieger, P. J. Meier and K. L. Brouwer, Biliary excretion in
primary rat hepatocytes cultured in a collagen-sandwichconfiguration,
Am. J. Physiol., 1999, 277, G12–21.
10 G. W. Sandker, R. M. E. Vos, L. P. C. Delbressine, M. J. H. Slooff,
D. K. F. Meijer and G. M. M. Groothuis, Metabolism of three
pharmacologically active drugs in isolated human and rat hepatocytes:
analysis of interspecies variability and comparison with metabolism in
vivo, Xenobiotica, 1994, 24, 143–155.
11 J. Sahi, Use of in vitro transporter assays to understand hepatic and
renal disposition of new drug candidates, Expert Opin. Drug Metab.
Toxicol., 2005, 1, 409–427.
12 J. V. Castell, R. Jover, C. P. Martinez-Jimenez and M. J. Gomez-
Lechon, Hepatocyte cell lines: their use, scope and limitations in drug
metabolism studies, Expert Opin. Drug Metab. Toxicol., 2006, 2, 183–
13 M. J. Gomez-Lechon, J. V. Castell and M. T. Donato, Hepatocytes-the
choice to investigate drug metabolism and toxicity in man: In vitro
variability as a reflection of in vivo, Chem. Biol. Interact., 2007, 168,
14 L. S. Smith and R. B. Allen, Bile reabsorption in rainbow trout
glycocholic acid, Ichthyology, 1999, 363–368.
15 H. Bonge, S. Hallen, J. Fryklund and J. E. Sjostrom, Cytostar-T
Bile Acid Uptake in Transfected HEK-293 Cells, Anal. Biochem., 2000,
16 K.-A. Ung, G. Olofsson, A. Fae, A. Kilander, C. Ohlsson and O.
Jonsson, In vitro determination of active bile acid absorption in
small biopsy specimens obtained endoscopically or surgically from the
human intestine, Eur. J. Clin. Invest., 2002, 32, 115–121.
17 J. Hardcastle, M. D. Harwood and C. J. Taylor, Absorption of
taurocholic acid by the ileum of normal and transgenic D F508 cystic
fibrosis mice, J. Pharm. Pharmacol., 2004, 56, 445–452.
18 C. D. Schteingart, S. Eming, H. T. Ton-Nu , D. L. Crombie and A. F.
Hofmann, Synthesis, structure, and transport properties of fluorescent
derivatives of conjugated bile acids, in Bile acids and the hepatobiliary
system, ed. G. Paumgartner, W. Gerok and A. Stiehl, London, Kluwer
Academic, 1992, pp. 177–183.
19 A. Benedetti, L. Marucci, A. Di Sario, G. Svegliati, Baroni, C. D.
Schteingart, H-T. Ton-Nu and A. F. Hofmann, Serial quantitative
image analysis of uptake and transport of fluorescent bile acids in
polarized biliary epithelial cells, Falk Symposium, 1995, 80, 191–194.
20 L. M. Maglova, A. M. Jackson, X-J. Meng, M. W. Carruth, C. D.
Schteingart, H-T. Ton-Nu, A. F. Hofmann and S. A. Weinman,
Transport characteristics of three fluorescent conjugated bile acid
analogs in isolated rat hepatocytes and couplets, Hepatology, 1995,
21 F. Holzinger, C. D. Schteingart, H. T. Ton-Nu, S. A. Eming,
M. J. Monte, L. R. Hagey and A. F. Hofmann, Fluorescent bile
acid derivatives: relationship between chemical structure and hepatic
and intestinal transport in the rat, Hepatotology, 1997, 26, 1263–
H-Z. Yeh and A. F. Hofmann, Transport of fluorescent bile acids by
the isolated perfused rat liver: kinetics, sequestration and mobilization,
Hepatology, 1998, 28, 510–520.
23 L. R. Hagey, C. D. Schteingart, S. S. Rossi, H-T. Ton-Nu and A. F.
Hofmann, An N-acyl glycyltaurine conjugate of deoxycholic acid in
the biliary bile acids of the rabbit, J. Lipid Res., 1998, 39, 2119–
cotransporting polypeptide (SLC10A1) and bile salt export pump
Drug Metab. Dispos., 2006, 34, 1575–1581.
microscopy, Photochem. Photobiol. Sci., 2005, 4, 13–22.
Visser, Lipid domains and rafts studied by time-resolved fluorescence
microspectroscopy, Biochem. Biophys. Lipids, 2006, 31–62.
two-dimensional distribution measurement of fluorescence lifetime,
Method Enzymol., 2006, 414, 633–642.
28 K. Hanaoka, K. Kikuchi, S. Kobayashi and T. Nagano, Time-resolved
long-lived luminescence imaging method employing luminescent lan-
29 S. Denicke, J-E. Ehlers, R. Niesner, S. Quentmeier and K-H. Gericke,
Steady-state and time-resolved two-photon fluorescence microscopy:
a versatile tool for probing cellular environment and function, Phys.
Scripta, 2007, 76, C115–C121.
30 J. B. Edel,P.Lahoud,A. E.G. Cass and A.J. De Mello,Discrimination
between single escherichia coli cells using time-resolved confocal
spectroscopy, J. Phys. Chem. B, 2007, 111, 1129–1134.
on cellular photosensitization: correlation of phototoxicity mechanism
with transient absorption spectroscopy measurements, Photochem.
Photobiol., 1998, 68, 51–62.
This journal is © The Royal Society of Chemistry and Owner Societies 2008Photochem. Photobiol. Sci., 2008, 7, 860–866 | 865
32 K. M. Bhattarai, A. P. Davis, J. J. Perry, C. J. Walter, S. Menzer and
D. J. Williams, A new generation of “cholaphanes”: steroid-derived
macrocyclic hosts with enhanced solubility and controlled flexibility,
J. Org. Chem., 1997, 62, 8463–8473.
and properties of fluorescent glycosamino-glycuronans labeled with 5-
aminofluorescein, Carbohydr. Res., 1982, 105, 69–85.
34 M. J. Gomez-Lechon, R. Jover, T. Donato, X. Ponsoda, C. Rodriguez,
K. G. Stenzel, R. Klocke, D. Paul, I. Guillen, R. Bort and J. V. Castell,
Long-term expression of differentiated functions in hepatocytes cul-
tured in three-dimensional collagen matrix, J. Cell. Physiol., 1998, 177,
35 G. Herrera, L. Diaz, A. Martinez-Romero, A. Gomes, E. Villam´ on,
namic approach to cell research, Toxicol. In Vitro, 2007, 21, 176–182.
36 M. F. Choi and P. Hawkins, Investigation of the response of dye-
nonaqueous solvent solutions to carbon dioxide, Anal. Chim. Acta,
1995, 309, 27–34.
37 M. F. Choi and P. Hawkins, A novel oxygen and/or car-
bon dioxide-sensitive optical transducer, Talanta, 1995, 42, 483–
38 C. Munkholm, D-R. Parkinson and D. R. Walt, Intramolecular
fluorescence self-quenching of fluoresceinamine, J. Am. Chem. Soc.,
1990, 112, 2608–2612.
39 M. Garcia-Escarp, V. Martinez-Munoz, I. Sales-Pardo, J. Barquinero,
J. C. Domingo, P. Marin and J. Petriz, Flow cytometry-based ap-
proach to ABCG2 function suggests that the transporter differentially
handles the influx and efflux of drugs, Cytometry A, 2004, 62, 129–
40 G. Juan, M. Cavazzoni, G. T. S´ aez and J. E. O’Connor, A fast kinetic
method for assessing mitochondrial membrane potential in isolated
41 G. Juan, R. C. Callaghan, R. Gil-Benso and J. E. O’Connor, Oxidative
metabolism in rat hepatoma and isolated hepatocytes: A comparative
flow cytometric study, Hepatology, 1996, 24, 385–390.
866 | Photochem. Photobiol. Sci., 2008, 7, 860–866This journal is © The Royal Society of Chemistry and Owner Societies 2008