Many aquatic organisms express the multixenobiotic resistance mechanism (MXR) mediated by a membrane protein denominated P-glycoprotein (Pgp), which reduce the accumulation of xenobiotics by active transport to out of cells. In order to establish fluorescence microscopy as a quantitative method to monitor MXR activity, the kinetic of fluorescence decay emitted by rhodamine B (RB) was determined. Rhodamine B (1 at 1000 nmoles L-1) were spotted on silica gel plate and fluorescence decay recorded and determined as exposition time (ET, sec) using a fluorescence microscope’s photo sensor. The ET at zero time was obtained from a linear equation of plotting ET(s) against the respective rhodamine B concentrations. The resulting mathematical model (RB = (28/ET) -1 -Blank, r2 = 0.9945), allowed the quantitative determination of rhodamine B intracellular accumulation (nmoles L-1This transport activity quantitation is consequence of the MXR mechanism activity operating in viable sections of gills of the mussel Perna perna and allows its measurement as a molecular biomarker

Fontana, José Domingos

Pessatti, Marcos Luiz

Portal de Periódicos da Univali (Universidade do Vale do Itajaí)

Ecotoxicol. Environ. Contam., v. 8, n. 1, 2013, 101-104
doi: 10.5132/eec.2013.01.014
Multixenobiotic resistance mechanism monitoring: standardization 
of fluorescence emmited by Rhodamine B
M.L. Pessatti1, 2 * &  J.D. Fontana3
1Universidade do Vale do Itajaí, Centro de Ciências Tecnológicas da Terra e do Mar (UNIVALI – CTTMar). 
CEP 88302-202.  Itajaí – SC, Brasil.
2 Universidade Federal do Paraná, Departamento de Bioquímica, Curitiba – PR, Brasil.
3 Universidade Tecnológica Federal do Paraná -  UTFPR - Ecoville, Curitiba – PR, Brasil.
(Received April 24, 2012; Accept May 06, 2013)
Abstract 
Many aquatic organisms express the multixenobiotic resistance mechanism (MXR) mediated by a membrane protein 
denominated P-glycoprotein (Pgp), which reduce the accumulation of xenobiotics by active transport to out of cells. In order 
to establish fluorescence microscopy as a quantitative method to monitor MXR activity, the kinetic of fluorescence decay 
emitted by rhodamine B (RB) was determined. Rhodamine B (1 at 1000 nmoles L-1) were spotted on silica gel plate and 
fluorescence decay recorded and determined as exposition time (ET, sec) using a fluorescence microscope’s photo sensor. The 
ET at zero time was obtained from a linear equation of plotting ET(s) against the respective rhodamine B concentrations. The 
resulting mathematical model (RB = (28/ET) -1 -Blank, r2 = 0.9945), allowed the quantitative determination of rhodamine 
B intracellular accumulation (nmoles L-1This transport activity quantitation is consequence of the MXR mechanism activity 
operating in viable sections of gills of the mussel Perna perna and allows its measurement as a molecular biomarker. 
Keywords: MXR, P-glycoprotein, fluorescence, P. perna, mussel, rhodamine.
Monitoramento do mecanismo de resistência a multixenobióticos: padronização da fluorescência emitida pela 
rodamina B
Resumo
Muitos organanismos aquáticos expressam o mecanismo de resistência à multixenobióticos (MXR) mediado pela proteína 
de membrana denominada glicoproteína-P (Pgp), a qual reduz o acúmulo de xenobióticos por transporte ativo para fora da 
célula. Com o objetivo de se estabelecer a microscopia como um método quantitativo para monitorar a atividade do mecanismo 
MXR, a cinética de decaimento da fluorescência emitida pela rodamina B (RB) foi determinada. Soluções de RB (1 a 1000 
nmoles L-1) foram aplicadas sobre uma placa de sílica gel e as intensidades de fluorescência ao longo do tempo registradas e os 
resultados expressos como tempo de exposição - ET (segundos) marcados pelo fotosensor de um microscópio de fluorescência. 
O ET no tempo zero foi obtido de uma equação linear do plote dos ET(s) com as respectivas concentrações de RB. O modelo 
matemático resultante (RB = (28/ET) -1 -Branco, r2 = 0.9945), permitiu a determinação quantitativa do acúmulo intracelular 
de rodamina B (nmoles L-1). Esta atividade de transporte é consequência da atividade do mecanismo MXR operando em 
fragmentos viáveis de brânquias do mexilhão Perna perna e permite seu uso como biomarcador molecular.
Palavras-chave: MXR, glicoproteína-P, fluorescência, P. perna, mexilhão, rodamina.
*Corresponding author: Marcos Luiz Pessatti; e-mail: pessatti@univali.br
102   Ecotoxicol. Environ. Contam., v.8, n. 1, 2013 Pessatti & Fontana
INTRODUCTION
The multidrug resistance (MDR) mechanism has been 
verified in many mammalian tumoral cell lines (Cornwall 
et al., 1995; Marzac et al., 2011; Latorre et al., 2012) and it 
is the main responsible for cell cross-resistance to multiple 
cytotoxic agents such as drugs utilized in chemotherapy 
(Gottesman et al., 1993; Steinbach & Legrand, 2007). This 
mechanism is mediated by a protein family, belonging to ATP-
binding cassette (ABC) transporters, which pumps (effluxes) 
xenobiotics out of cells in an ATP-dependent manner. Among 
these proteins, the P-glycoprotein (Pgp) and multidrug 
resistance-associated proteins (MRPs) act as transporters in 
secretory processes, determining the cellular permeability to 
different structurally unrelated compounds (e.g., cytotoxic, 
xenobiotics), enhancing the cell resistance to these agents. 
Many aquatic organisms express the phenotype of 
multixenobiotic resistance (MXR) mechanism in a manner 
analogue to the multidrug resistance (MDR) mechanism 
(Kurelec & Pivcevic, 1991; Loncar et al., 2010). The 
MXR bestow to these animals the capacity of adaptation 
to contaminated environments. As it is inherently present 
in all tested aquatic organisms, the MXR mechanism 
has been considered as a natural biological ‘first line’ 
mechanism of defense of protection against xenobiotics 
(Kurelec et al., 1992). 
The activity of MXR mechanism has been quantified 
by different methods, involving binding assays utilizing 
radioactive isotopes (Simmons et al., 1995; Kurelec et al., 
1997) and fluorescence measurements (Broxterman et al., 
1997; Pessatti et al., 2002; Zaja et al., 2011) irrespective to the 
cells or organisms origin. Rhodamine B is an useful probe as it 
is recognized as a substrate by Pgp and allows, using activity 
transport assays, the measurement of the MXR mechanism 
activity. However, the relationship between the rhodamine B 
concentration and fluorescence intensity is fundamental for 
the consolidation of fluorescence microscopy as a quantitative 
method of MXR mechanism activity. This approach has been 
used in fresh gill sections of the mussel Perna perna in order 
to monitor the transport (accumulation and efflux) of the 
fluorescent dye through in situ experiments. 
MATERIAL AND METHODS
Purification of rhodamine B
To the purification of rhodamine B P.A. (Merk) was used 
a conventional chromatographic procedure. The rhodamine B 
(250 mg) was fractionated in a column of silica gel (Merk) with 
20 mL (total volume) and eluted with chloroform: methanol 
(9:1). Fractions of 8 mL were collected and monitored by thin 
layer chromatography. The fractions 2-6 were assembled and 
rechromatographed in same system. The fractions 1 and 2, 
containing the pure rhodamine B, were concentrated under 
vacuum.
Standardization of fluorescence emitted by rhodamine B
To the experiments an eppifluorescence microscope 
Olympus BX60 equipped with U-MWG filter (excitation and 
emission wavelengths of 510-550 and 590 nm respectively) 
was used. The fluorescence emission with the closed 
diaphragm from the stained area was quantified by a coupled 
photo sensor Olympus PM20 regulated at ISO 100 and with 
a 4.0 exposition adjustment (maximum sensitivity), according 
Pessatti et al. (2002).
To the standardization of fluorescence emitted by 
rhodamine B, spots of 10 uL of purified rhodamine B (from 
1 to 1000 nmoles L-1) were carefully applied on silica gel (60 
F 254, Riedel-de Haën) plates in order to generate uniform 
circular areas. The kinetics of fluorescence decay was then 
recorded from 0 to 90 seconds using the Exposition Time (ET) 
recorded in the fluorescence microscope’s photo sensor as a 
measurement unit. ET was defined as the necessary time, in 
seconds, of shutter opening to sensitize a photographic film 
under the condition described above. A calibration curve 
was built and the zero time (maximum fluorescence) of each 
rhodamine B concentration obtained from linear equations of 
ET plotted against the respective rhodamine B concentrations. 
The determinations were performed in triplicate and the 
results represent the mean of independent experiments (from 
2 to 6), with the respective confidence intervals (α=0.05). As a 
control, the same procedure was performed in silica gel plates 
spotted with the same volume of distilled water, since it was 
the solvent used for the dye solubilization.
Assay of rhodamine B transport
In order to verify the standardization of fluorescence, 
different assays were assembled using gills of the mussel Perna 
perna. The mussel specimens (50-70 mm long) were collected 
at the Penha Experimental Sea Culture Station, UNIVALI - SC, 
Brazil, and were acclimatized in laboratory during 24 hours. 
Rhodamine B accumulation experiments were performed 
following Cornwall et al. (1995), with some modifications. The 
mussels were carefully dissected and the gills were collected in 
filtrated sea water. Small sections (2.5 mm2) were cut from the 
dorsal edges of the descending arms of each lamella, above the 
interlamellar tissue bridges, and were incubated in sea water 
with aeration during 20 minutes for mucus removal. The gill 
sections were then re-incubated with 2 mL of 1 µmoles L-1 
(or less) rhodamine B in sea water, for 15 minutes at 16o C, 
followed by a 20 sec rinse with sea water in order to discard any 
extracellular residual dye. Each gill piece was then assembled on 
a microscope plate. The fluorescence intensity of the abfrontal 
side of each gill filament was measured in the fluorescence 
microscope as described. Control for any basal fluorescence 
was obtained from gill sections incubated in the absence of 
rhodamine. The fluorescence from fifteen to thirty filaments 
from each gill section was/were recorded. Final results of the 
recorded ETs were then expressed as averages and confidence 
intervals (α=0.05) of the rhodamine B accumulation (nmoles L-1) 
in the gill tissue, calculated respectively from standardization 
described above. This unit (nmoles L-1) should be understood 
Ecotoxicol. Environ. Contam., v.8, n. 1, 2013   103Rhodamine B to MXR studies
as the mass of rhodamine B accumulated in tissue that give a 
fluorescence intensity equivalent at this concentration when 
observed in silica gel plate.  
RESULTS
The kinetics of fluorescence decay emitted by purified 
rhodamine B as function of UV light exposition time is 
shown in the insert of Figure 1. The ETs at zero times (T0) 
were estimated from the linear equations (mean r2=0.9946) 
obtained by plotting ETs at several times of UV exposition 
versus the respective concentrations for rhodamine B. Means 
and confidence intervals (α=0.05) from 2 to 6 independent 
assays are shown.
Because of the negative exponential profile obtained, many 
mathematic models were tried to explain the observed behavior. 
The chosen model to relate ET recorded by the eppifluorescence 
microscopy and rhodamine B concentration was:
BlankETRB −−= 1)/28(                          (Equation 1)
where RB is the rhodamine B concentration (in nmoles 
L-1), ET is the exposition time (in seconds) and Blank is the 
exposition time (in seconds) verified in mussel gills incubated 
with only sea water (basal fluorescence) (Fig. 2).  The r2 to this 
model was 0.9945.
DISCUSSION
The multixenobiotic resistance mechanism is a ‘first 
line’ of defense against numerous compounds recognized as 
xenobiotic by cells (Kurelec, 1992). However, to study the 
MXR in in vivo systems is necessary methods that enable the 
quantitative determination of transport activity of standard 
substrates. The rhodamine B is an excellent fluorophore, since 
is water soluble, is widely used for many purposes (what 
make it easily available) and has a low cost. Moreover, is a 
good substrate of Pgp and other ABC transporters (Smital et 
al., 2003; Nabekura et al., 2010; Faria et al., 2011).  
As can be observed, the rhodamine B concentration can 
be calculated as zero at ET=28 seconds and negative values 
will be obtained if ET>28 seconds (a situation seldom 
observed experimentally), defining the sensibility limit of the 
method. Nevertheless, this mathematical model shows a good 
correlation with ET values as low as 0.03 seconds, which 
represents 1000 nmoles L-1 of rhodamine B, with an estimated 
error of 5.83%. The ETs verified in in situ experiments 
were related with the rhodamine B concentration(s). Hence, 
Equation 1 allows the quantitative determination of the MXR 
mechanism activity in in situ experiments.
The results obtained from in situ assays demonstrate 
the usefulness of this approach as a quantitative method for 
measuring rhodamine B in live tissues, and it can be used as 
expression of MXR mechanism in mussel Perna perna, as 
well as in other organisms.
REFERENCES
BROXTERMAN, H.J., SCHUURHUIS, G.J., LANKELMA, J., 
OBERINK, J.W., EEKMAN, C.A., CLAESSEN, A.M.E., 
HOEKMAN, K., POOT, M. & PINEDO, H.M., 1997, Highly 
sensitive and specific detection of P-glycoprotein function for 
haematological and solid tumour cells using a novel nucleic acid 
stain.  Br. J. Cancer, 76:1029-1034. http://dx.doi.org/ 10.1038/
bjc.1997.503
CORNWALL, R., TOOMEY, B. H., BARD, S., BACON, C., 
JARMAN, W.M. & EPEL, D., 1995, Characterization of 
Figure 2 - Saturation of multixenobiotic resistance (MXR) mechanism 
by purified rhodamine B. Sections gills of mussel P. perna were incubated 
in 2 mL of rhodamine B (from 10 nmoles L-1 to 10 µmoles L-1) by 30 
minutes, washed in sea water to remove any extracellular dye, and then 
placed on slides for measuring fluorescence intensity.
Figure 1 - Calibration curve of fluorescence emitted by purified 
rhodamine B. Exposition time(s) (ET, seconds) were obtained from 
different purified rhodamine B concentrations applied to silica gel and 
exposed to the microscope´s UV light at zero time (mean ± confidence 
interval (p<0.05) of 2 from 6 independent assays). Insert: the kinetics of 
fluorescence decay emitted by purified rhodamine B applied to silica gel 
when excited by microscope´s UV light. ETs mean ± confidence interval 
(p<0.05) obtained from 1 nmoles L-1 (), 2 nmoles L-1 (∆), 3 nmoles L-1 
(), 4 nmoles L-1 (), 10 nmoles L-1 (), 30 nmoles L-1 (▲) and 100 
nmoles L-1 () purified rhodamine B exposed to UV light in time (0 at 90 
seconds) in epifluorescence microscope. The results show one of many 
(from 2 to 6) independent assays, with r2 of 0,9946 (mean of all assays).
B = (28/ T) -1 - Blank
104   Ecotoxicol. Environ. Contam., v.8, n. 1, 2013 Pessatti & Fontana
multixenobiotic/multidrug transport in the gills of the mussel 
Mytilus californianus and identification of environmental 
substrates. Aquat. Toxicol., 31:277-296. http://dx.doi.
org/10.1016/0166-445X(94)00070-7
CORNWELL, M. M., GOTTESMAN, M.M. & PASTAN, I.H., 
1986, Increased vinblastine binding to membrane vesicles from 
multidrug-resistant KB cells. J. Biol. Chem., 261:7921-7928.
FARIA, M., NAVARROA, A., LUCKENBACHB, T., PIÑA, B. 
& BARATA, C., 2011, Characterization of the multixenobiotic 
resistance (MXR) mechanism in embryos and larvae of the zebra 
mussel (Dreissena polymorpha) and studies on its role in tolerance 
to single and mixture combinations of toxicants. Aquat. Toxicol., 
101:78–87. http://dx.doi.org/10.1016/j.aquatox.2010.09.004
GOTTESMAN, M.M. & PASTAN, I., 1993, Biochemistry of 
multidrug resistance mediated by the multidrug transporter. Ann. 
Rev. Biochem., 62:385-427. http://dx.doi.org/10.1146/annurev.
bi.62.070193.002125
NABEKURA, T., YAMAKIA, T., HIROIA, T., UENOA, K. & 
KITAGAWAB, S., 2010, Inhibition of anticancer drug efflux 
transporter P-glycoprotein by rosemary phytochemicals. 
Pharmacol. Res., 61:259–263. http://dx.doi.org/10.1016/j.
phrs.2009.11.010
KURELEC, B. & PIVCEVIC, B., 1991, Evidence for a 
multixenobiotic resistance mechanism in the mussel Mytilus 
galloprovincialis. Aquat. Toxicol., 19:291-302.KURELEC, B., 
KRCA, S., PIVCEVIC, B., UGARKOVIC, D., BACHMANN, 
M., IMSIECKE, G. & MUELLER, W.E.G., 1992, Expression 
of P-glycoprotein gene in marine sponges. Identification and 
characterization of the 125 kDa drug-binding glycoprotein. 
Carcinogenesis, 13:69-76.KURELEC, B., SMITAL, T., 
BRITVIC, S., PIVCEVIC, B., KRCA, S., JELASKA, 
S.B., SAUERBORN R. & MUSTAJBEGOVIC, S., 1997, 
Multixenobiotic defence mechanism in aquatic organisms. 
Period. Biol., 99:319-328.
LATORRE, E., TEBALDI, T., VIERO, G., SPARTA, A.M., 
QUATTRONE, A. & PROVENZANI A., 2012, Downregulation 
of HuR as a new mechanism of doxorubicin resistance in 
breast cancer cells. Mol. Cancer, 11(1):13. http://dx.doi.
org/10.1186/1476-4598-11-13
LONCAR, J., POPOVIĆ, M., ZAJA, R. & SMITAL, T., 2010, 
Gene expression analysis of the ABC efflux transporters in 
rainbow trout (Oncorhynchus mykiss). Comp. Biochem. 
Physiol. C Toxicol. Pharmacol., 151(2):209-15. http://dx.doi.
org/10.1016/j.cbpc.2009.10.009
MARZAC, C., GARRIDO, E., TANG, R., FAVA, F., HIRSCH, P., DE 
BENEDICTIS, C., CORRE, E., LAPUSAN, S., LALLEMAND, 
J.Y., MARIE, J.P., JACQUET, E. & LEGRAND, O., 2011, ATP 
Binding Cassette transporters associated with chemoresistance: 
transcriptional profiling in extreme cohorts and their prognostic 
impact in a cohort of 281 acute myeloid leukemia patients. 
Haematologica, 96(9):1293-301. http://dx.doi.org/10.3324/
haematol.2010.031823
PESSATTI, M. L., RESGALLA JR., C., REIS FO, R.W., KUEHN, 
J. & FONTANA, J.D., 2002, Variability of rates of filtration, 
respiration and assimilation and of multixenobiotic mechanism 
resistance (MXR) of mussel Perna perna under lead influence. 
Braz. J. Biol., 62(4A):651-656.  http://dx.doi.org/10.1590/S1519-
69842002000400013
SIMMONS, N.L., HUNTER, J. & JEPSON, M.A., 1995, Targeted 
delivery of a substrate for P-glycoprotein to renal cysts in vitro. 
Biochem. Biophys. Acta, 1237:31-36.
SMITAL, T., SAUERBORN, R. & HACKENBERGER, B.K., 2003, 
Inducibility of the P-glycoprotein transport activity in the marine 
mussel Mytilus galloprovincialis and the freshwater mussel 
Dreissena polymorpha. Aquat. Toxicol., 65:443–465. http://
dx.doi.org/10.1016/S0166-445X(03)00175-9
ZAJA, R., LONČAR, J., POPOVIC, M. & SMITAL, T., 2011, 
First characterization of fish P-glycoprotein (abcb1) substrate 
specificity using determinations of its ATPase activity and calcein-
AM assay with PLHC-1/dox cell line. Aquat. Toxicol., 103(1-
2):53-62. http://dx.doi.org/10.1016/j.aquatox.2011.02.005


English

Multixenobiotic resistance mechanism monitoring: standardization of fluorescence emmited by Rhodamine B

https://siaiap32.univali.br/seer/index.php/eec/article/download/3692/2557

Multixenobiotic resistance mechanism monitoring: standardization of fluorescence emmited by Rhodamine B

Abstract

Similar works

Full text

Available Versions

Portal de Periódicos da Univali (Universidade do Vale do Itajaí)

Portal de Periódicos da Univali (Universidade do Vale do Itajaí)