Activation of multidrug efflux transporter activity at fertilization in sea urchin embryos (Strongylocentrotus purpuratus). Dev Biol

Hopkins Marine Station, Stanford University, 120 Oceanview Boulevard, Pacific Grove, CA 93950, USA.
Developmental Biology (Impact Factor: 3.55). 01/2005; 276(2):452-62. DOI: 10.1016/j.ydbio.2004.09.013
Source: PubMed
ABSTRACT
This study presents functional and molecular evidence for acquisition of multidrug transporter-mediated efflux activity as a consequence of fertilization in the sea urchin. Sea urchin eggs and embryos express low levels of efflux transporter genes with homology to the multidrug resistance associated protein (mrp) and permeability glycoprotein (p-gp) families of ABC transporters. The corresponding efflux activity is low in unfertilized eggs but is dramatically upregulated within 25 min of fertilization; the expression of this activity does not involve de novo gene expression and is insensitive to inhibitors of transcription and translation indicating activation of pre-existing transporter protein. Our study, using specific inhibitors of efflux transporters, indicates that the major activity is from one or more mrp-like transporters. The expression of activity at fertilization requires microfilaments, suggesting that the transporters are in vesicles and moved to the surface after fertilization. Pharmacological inhibition of mrp-mediated efflux activity with MK571 sensitizes embryos to the toxic compound vinblastine, confirming that one role for the efflux transport activity is embryo protection from xenobiotics. In addition, inhibition of mrp activity with MK571 alone retards mitosis indicating that mrp-like activity may also be required for early cell divisions.

Full-text

Available from: Amro Hamdoun
Activation of multidrug efflux transporter activity at fertilization in
sea urchin embryos (Strongylocentrotus purpuratus)
Amro M. Hamdoun
a,
*
, Gary N. Cherr
b,c
, Troy A. Roepke
c
, David Epel
a
a
Hopkins Marine Station, Stanford University, Pacific Grove, CA 93950, United States
b
Departments of Environmental Toxicology and Nutrition, University of California, Davis, CA 95616, United States
c
Bodega Marine Laboratory, UC Davis, Bodega Bay, CA 94923, United States
Received for publication 8 August 2004, revised 8 September 2004, accepted 9 September 2004
Available online 14 October 2004
Abstract
This study presents functional and molecular evidence for acquisition of multidrug transporter-mediated efflux activity as a consequence
of fertilization in the sea urchin. Sea urchin eggs and embryos express low levels of efflux transporter genes with homology to the multidrug
resistance associated protein (mrp) and permeability glycoprotein (p-gp) families of ABC transporters. The corresponding efflux activity is
low in unfertilized eggs but is dramatically upregulated within 25 min of fertilization; the expression of this activity does not involve de novo
gene expression and is insensitive to inhibitors of transcription and translation indicating activation of pre-existing transporter protein. Our
study, using specific inhibitors of efflux transporters, indicates that the major activity is from one or more mrp-like transporters. The
expression of activity at fertilization requires microfilaments, suggesting that the transporters are in vesicles and moved to the surface after
fertilization. Pharmacological inhibition of mrp-mediated efflux activity with MK571 sensitizes embryos to the toxic compound vinblastine,
confirming that one role for the efflux transport activity is embryo protection from xenobiotics. In addition, inhibition of mrp activity with
MK571 alone retards mitosis indicating that mrp-like activity may also be required for early cell divisions.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Multidrug efflux; Fertilization; Embryo; mrp; p-gp; Activation; psc833; mk571; Calcein-am; SpABCB1; SpABCC1; SpABCC2; Mitosis
Introduction
Fertilization results in physiological and structural
modification of the egg. In sea urchins, the changes in
egg activity result from a transient rise in calcium and
permanent rise in intracellular pH; these initiate structural
and enzymatic changes that ultima tely lead to initiation of
DNA synthesis and rapid cell divisions (Epel, 1990; Jaffe et
al., 2001; Runft et al., 2002). The structural changes center
on reorganization of embryonic surfaces, initially arising
from cortical granule exocytosis and formation of the
fertilization envelope. The exocytosis is followed by
endocytosis and later sequential movements of vesicles to
the cortex (Matese et al., 1997; Connor and Wessel, 1998;
Smith et al., 2000). Some of these vesicles contain
constituents of the extracellular matrix and their movement
is sensitive to cytochalasin and latrunculin suggesting that
the trafficking occurs on microfilaments (Ikebuchi et al.,
2001; Wessel et al., 2002). It is also hypothesized that some
of these vesicles contain amino acid transporters that are
similarly inserted into the plasma membrane and then used
to bring in amino acids from the surrounding seawater
(Epel, 1972; Schneider, 1985; Wright and Manahan, 1989).
Here, we show that the fertilization response involves
upregulation of multidrug efflux transport activity, as part of
the re-organization of the cytoplasm and plasma membrane.
This activity protects the embryo from exogenous toxicants
and inhibition of the efflux activity affects cell division,
suggesting that the transporter activity also has some role in
passage through mitosis.
These multidrug (mdr) efflux transporters are members of
the ATP Binding Cassette (ABC) super-family and actively
0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2004.09.013
* Corresponding author. Hopkins Marine Station, Stanford University,
120 Oceanview Boulevard, Pacific Grove, CA 93950.
E-mail address: hamdoun@stanford.edu (A.M. Hamdoun).
Developmental Biology 276 (2004) 452 462
www.elsevier.com/locate/ydbio
Page 1
export or sequester endogenous and exogenous compounds
(Borst et al., 2000; Dean et al., 2001; Litman et al., 2001)
including various xenobiotics ( Leslie et al., 2001). The
xenobiotic transporters include permeability glycoproteins
(p-gp; Ambudkar et al., 1999), multidrug resistance associ-
ated proteins (mrp; Cole and Deeley, 1998) and White family/
mitoxantrone (mxr) resistance prote ins (Litman et al., 2000).
In addition to providing protection by xenobiotic transport,
these proteins regulate cellular and developmental physiol-
ogy by efflux of endogenous signaling molecules. In
Dictyostelium, the ABC transporter RhT regulates terminal
stalk cell differentiation by efflux transport of DIF-1, an
endogenously produced differentiation factor (Good and
Kuspa, 2000). Efflux transporters are also involved in
establishing the auxin gradients required for plant develop-
ment (Friml et al., 2003; Gaedeke et al., 2001).
Most attention has focused on the protective role of these
transporters in somatic cells but they are also imp ortant in
embryo protection. For example both, embryos and adults of
the marine echiur oid worm Urechis caupo express a p-gp
transporter which protects them from naturally occurring
microbial toxins (Hamdoun et al., 2002; Toomey and Epel,
1993; Toomey et al., 1996). Embryos of Caenorhabditis
elegans express p-gp and mrp genes that are associated with
tolerance to heavy metals (Broeks et al., 1996). Similarly
mouse embryos have an efflux transport phenotype thought
to be mediated by a p-gp transporter and which protects the
embryo from xenotoxins (Elbling et al., 1993).
Previous work had suggested that early sea urchin
embryos and larvae do not have p-gp-mediated toxicant
defenses (Hamdoun et al., 2002; Toomey and Epel, 1993),
although the adults can live in poll uted environments. Here,
we show that several mdr transporter-like g enes are
expressed in the emb ryo, including at least one with
homology to p-gp and two with homology to mrps. The
major efflux activity in the sea urchin egg is mrp-like and this
activity is dramatically upregulated withi n 25 min of
fertilization. This increase does not involve de novo tran-
scription or translation, but rather the transporter proteins are
present in eggs and their activity increases by a microfila-
ment-dependent process, most likely involving movement of
transporter vesicles to the egg surface and insertion. We show
that this transport activity protects the embryo from toxic
xenobiotic compounds such as vinblast ine. Finally, inhib-
ition of mrp-like transport activity slows progression through
mitosis with a major effect on progression through anaphase.
This suggests that mrp-mediated transport of some endog-
enous substance may also be required for early cell divisions.
Materials and methods
Animals
Purple sea urchins, Strongylocentrotus purpuratus, were
kept in flow through sea wat er tanks (approximately 138C)
and fed kelp. Gametes were collected by intracoelomic
injection with 0.5M KCl. Eggs were washed several times in
1.0 Am filtered seawater (FSW) prior to use in experiments.
For individual embryo transport measurements, embryos
were prepared by mixing eggs from 2 to 4 fema les, with
diluted sperm from one mal e. Only batches of embryos with
N90% successful development (assessed at the two-cell
stage) were used for experiments. All experiments were
conducted at 148C.
Fluorophores and inhibitors
MK571 was obtained from Cayman Chemicals. PSC833
was a gift from Novartis (Basel, Switzerland). Reversin
(R205) was initially obtained by gift of Dr. Balzas Sarkadi
and later purchased from Sigma (St. Lois, MO). Actino-
nmycin-D, emetine and verapamil were also purchased from
Sigma. Calcein-acetoxymethylester (c-am), cytochalasin-D
and latrunculin-A were obtained from Calbiochem. Sytox-
green was from Molecular Probes (Eugene, OR). DMSO was
used as a solvent for all stocks. Chemical stocks were
prepared at approximately 1000
the final desir ed concen-
tration such that highest concentration of DMSO did not
exceed 0.5% of the experimental volume; this solvent
concentration has no effect on transport activity or activation
of transport.
Degenerate PCR and cDNA library screening
Packed eggs or embryos were flash frozen in liquid
nitrogen, homogenized in a liquid nitrogen cooled ceramic
mortar, and then thawed into 10 volumes of homogenization
buffer (Kalinowski et al., 2003 ) containing proteinase K (200
Ag/ml). Homogenates were incubated for 45 min at 378C
before phenol-chloroform extraction and ethanol precipita-
tion. RNA was further purified by 8 M lithium chloride
precipitation ( Sambrook and Russell, 2001). cDNA was
synthesized with an oligo-dT 12–18 primer and Superscript
Reverse Transcriptase (Invitrogen, Carlsbad, CA) according
to manufacturers instructions. Degenerate primers (Al lik-
mets and Dean, 1998; Lee et al., 1998) were used to amplify
conserved fragments of the corresponding transporter
cDNAs by RT-PCR and clone a heterologous cDNA
fragment containing a conserved 230-bp region of sea star
(Asterina miniata) transporter cDNA; 5VTCGGGATC-
CAGGGAGAATATACTGTTTGGGGCACCATTGCAA-
GATGCAGAGTACCAGCGAGTGATCGAAGCC-
TGCGCGCTGGCCCCTGATCTGGACATGCTCC-
CAGCTGGAGATCTAACGGAGATTGGAGAAAAGG-
GAATTAACCTGAGTGGTGGTCAGAAGCAGAGGGT-
CAGT TTAGCTCGAGCCGTTTACA ACAATGCCGACA-
TCTATCTCCTTGACGACGAATTCCG3V. This fragment
was random primed and used to screen cDNA macroarray
filter libraries of S. purpuratus (Cameron et al., 2000; Clark
et al., 1999) using standard filter hybridization techniques
(Sambrook and Russell, 2001).
A.M. Hamdoun et al. / Developmental Biology 276 (2004) 452–462 453
Page 2
cDNA characterization
Clones corresponding to positive coordinates on the
cDNA library screen were ob tained from cDNA libraries
maintained at Caltech as part of the Sea Urchin Genome
Project (SUGP). Clones were grown on selective plates and
individual colonies were selected for sequencing. cDNA
inserts (pSport vector; Invitrogen) were analyzed for size on
agarose gels and SP6 and T7 ends were sequenced (Davis
Sequencing, Davis CA) to confirm the identity of each
clone. All of the positive clones were completely sequenced
using a trans poson-based shotgun sequencing approach
(GPS, New England Biolabs; Beverly MA) on an ABI
3100 sequencer using standard cycle sequencing protocols.
Contiguous partial cDNA seq uences were e dited and
assembled using Sequencher. Sequencing coverage was at
least 2
for all sequences. The reading frame, homology
and sequence analyses of the putative proteins encoded by
these partial cDNAs were determined using tools at the
Expasy Molecular Biology Server (http://us.expasy.org/)
and at NCBI.
Real-time PCR
Real-time PCR was performed at the Lucy W hittier
molecular core facility, UC Davis according to the following
protocol. Fluorescent labeled TaqMan probe (5Vend, reporter
dye FAM (6-carboxyflourescein), 3V end, quencher dye
TAMRA (6-car boxytetramethylrhodamine)) were designed
using Primer Express software (Applied Biosystems, Foster
City, CA). SpABCB1 primer sequences were forward
5VGGAGCTTGAGAAGCATTTCAGC3Vand reverse 5VAA-
GGAGGATGAAACCACACTCTG3V with the probe
sequence 5VTGATGCGAGTCTGGAAG CTCAACACC3V.
SpABCC1 forward primer was 5VGATGGCGATGTAAC-
GGACAGT3V and rever se 5VTGATGGTCACCGAA A-
GATCGT3V with the probe 5VCCGAGAGCGTGGGTAA-
TTCACACGA3V. TaqMan PCR systems were evaluated
according to Leutenegger et al. (1999).
For q-pcr exp eriments, three separate batches of sea
urchin eggs and embryos were prepared for sample
collection. Samples were taken immediately after termi-
nation of the experiments and lysed in 1
ABI lysis
buffer (Applied Biosystems). Before RNA extraction, the
lysates were transferred into 96 deep well plat es and
digested with Proteinase K (Invitrogen). The total RNA
was extracted from the lysates using a 6700 automated
nucleic acid (ANA) workstation (Applied Biosystems)
according to manufacturers instructions. Total RNA was
eluted in 100 Al of elution buffer. Complementary DNA
(cDNA) was synthesized using 100 units of SuperScript
III (Invitrogen), 300 ng random hexadeoxyribonucleotide
(pd(N)
6
) primers (random hexamer primer) 10 U RNase-
Out (RNase inhibitor), and 1 mM dNTPs in a final
volume of 40 Al. Each PCR reaction contai ned 400 nM
of each primer, 80 nM of the TaqMan probe and
commercially available PCR mastermix (TaqMan Univer-
sal PCR Mastermix, Applied Biosystems) containing 10
mM Tris–HCl (pH 8.3), 50 mM KCl, 5 mM MgCl
2
, 2.5
mM deoxynucleotide triphosphates, 0.625 U Ampli Taq
Gold DNA polymerase per reaction, 0.25 U AmpErase
UNG per reaction and 5 Al of the diluted cDNA sample
in a final volume of 25 Al. The samp les were placed in
96 well plates and amplified in an automated fluorometer
(ABI PRISM 7700 Sequence Detection System, Applied
Biosystems). Amplification conditions were 2 min at
508C, 10 min at 958C, 40 cycles of 15 s at 95 8C and 60
sat608C.
Final relative quantitation was done using the compara-
tive cycles to threshold (C
T
) method (User Bulletin #2,
Applied Biosystems) and is reported as relative transcription
or the n-fold difference relative to eggs. The endogenous
control for the sea urchin samples 18S rRNA was used to
normalize the mrp TaqMan signals. The DC
T
values were
calibrated agains t the control DC
T
values for each target
gene. The relative linear amount of target mol ecules relative
to the calibrator, was calculated by 2
DDCT
.
Fluorescence microscopy
Accumulation of calcein was used to measure mrp trans-
porter activity in eggs and embryos. Calcein-AM (c-am) is a
non-fluorescent mrp and p-gp substrate. Eggs and embryos
are incubated in 250 nM c-am and any c-am that enters the cell
is rapidly hydrolyzed by esterases to form fluorescent,
membrane-impermeant calcein, which is detected as intra-
cellular fluorescence within minutes of extracellular c-am
addition (Essodaigui et al., 1998). Thus, low transport activity
results in high calcein accumulation and high fluorescence
while increased transporter activity results in reduced intra-
cellular accumulation of calcein and reduced fluorescence.
Visualization of DNA for cell division experiments was
performed by fixing embryos overnight in 2% formaldehyde
and 0.1% Tween 20. Fixative was washed out 2
in Tris-
buffered saline with 0.1% Tween (TBST). Embryos were
then resuspended in 10 nM Sytox green (Molecular Probes)
in TBST. DNA was visualized with FITC wavelength filters
(as described below).
Fluorescence imaging was performed using a fixed stage
upright microscope (Olympu s BX50-WI) with xenon
illumination and a cooled (148C) stage, or an inverted
microscope (Zeiss Axiovert S-100; Germany) equipped with
mercury illumination. Exc itation was set to 488 Dm using a
Lambda-10 automated filter wheel (Sutter Instruments,
Novato, CA). Images were captured using a Photometrics
Coolsnap FX cooled charge coupled device (CCD) camera
(Olympus BX50-WI; Tokyo, Japan) or a Princeton Instru-
ments cooled CCD camera (Zeiss S100). A 10
objective
was used for all physiology experiments except the time-
lapse recordings of eggs at fertilization which were collected
using a 20
objective. Pseudo color micrographs and the
visualization of DNA were performed with a 40
objective.
A.M. Hamdoun et al. / Developmental Biology 276 (2004) 452–462454
Page 3
Data analysis and presentation
Fluorescence intensity of individual eggs and embryos
was measured using image analysis macros provided with
MetaMorph (Universal Imaging Corp., Downingtown, PA)
software. All comparisons of changes in fluorescence
intensity between treatments were made from data corrected
for background (70 units). P values were computed from
two sample t tests of means.
Results
Identification of transporter genes in the egg
We confirmed that several efflux transporter mRNAs
are expressed in the sea urchin egg and we characterized
partial cDNAs encoding large fragments of three unique
mRNAs; SpABCB1 (1289AA; Genbank accession number
CK995664), encoding a p-gp, and large fragm ents of two
distinct mrp-like cDNAs, SpABCC1 (765AA; CK995665)
and SpABCC2 (709AA; CK995667). BlastP comparison of
the translated cDNAs on Genbank (NCBI) indicate highly
significant homology (E b 1
10
50
) to mammalian p-gp1,
mrp2, mrp5, and sulfonylureas (ABCC8/9). Each partial
mrp cDNA encoded about half of the expected protein with
at least one ABC transporter ATPase domain and one ABC
transporter type-1 integral transmembrane domain (Prosite).
The p-gp cDNA was nearl y full-length, as expect ed by
homology to other p-gps. Multiple alignment of the protein
sequences by ClustalW revealed that observed urchin
sequences align with other vertebrate and invertebrate
sequences consistent with their predicted BlastP identities
(Fig. 1).
Basis of efflux transport assay
We adapted the methods of Homolya et al. (1996) and
Essodaigui et al. (1998) which employ calcein-acetoxyme-
thylester (c-am) to measure mdr tran sporter mediated
transport activity (Essodaigui et al., 1998; Hollo et al.,
1994, 1996, 1998; Homolya et al., 1996). Calcein-AM is a
non-fluorescent compound that is a substrate of both mrp
and p-gp. If the cell has low p-gp or mrp activity, or no
activity, c-am accumulates in the cells and is rapidly
hydrolyzed by n on-specific esterases to form green,
fluorescent calcein. Unlike c-am, free calcein is relatively
membrane impermeant and therefore remains in the cell
(Essodaigui, 1998). If a cell has high levels of p-gp or mrp
transport activity, c-am is prevented from accumulating in
the cell by efflux transport and fluorescent calcein accum u-
lates to a lesser extent.
A specific test for multidrug transporter-mediated efflux
activity is to measure intracellular calcein accumulation in
the presence or absence of mdr inhibitors. If there is no or
little transport activity, incubation in a transport inhibitor has
little or no effect and there is no or little increased
fluorescence in the cell. In contrast, if there is transport
activity, incubation in an inhibitor results in increased
calcein accumulation and fluorescence as compared to the
fluorescence in the cell when no inhibitor is present.
Initiation of transport at fertilization
Fig. 2 compares the kinetics of calcein accumulation in
the presence of 250 nM extracellular c-am in eggs and in
2–4 cell embryos. Calcein accumulates in unfertilized eggs
in a linear fashion over time whereas in 2–4 cell embryos
intracellular calcein fluorescence is barely detectable even
after 2 h of incubation in c-am. The reduced calcein
accumulation in embryos relative to eggs could be a
consequence of reduction in calcein-AM permeability, a
reduction in intracellular esterase activity, or result from
increased expression of a calcein efflux transporter activity.
Fig. 3 shows that embryos incubated in the presence of the
mdr inhib itors cyclosporin-A, reversin-2 05, verapamil,
PSC833, or MK571 accumulate increased levels of calcein.
This indicates that the reduced accumulation in the
embryos largely results from increased efflux transport,
since the addition of the various mdr inhibitors results in
calcein accumulation.
We found that differences in passive c-am permeability
and esterase activity do not account for the changes in c-am
accumulation at fertilization since the absolute fluor escence
Fig. 1. Multiple sequence alignment (ClustalW) shows that the sea urchin multidrug transporter proteins and partial cDNAs segregate into three lineages
relative to human and invertebrate mdr proteins. SpABCB1 is a p-gp that aligns more closely with p-gp and mxr (a functional half transporter) than any of the
other proteins. The partial cDNAs SpABCC1 and SpABCC2 are both mrp-like and the former most similar to human mrp5 and 8, whereas the latter is closer to
the other invertebrate (C. elegans) mrps and human mrp2.
A.M. Hamdoun et al. / Developmental Biology 276 (2004) 452–462 455
Page 4
levels of embryos treate d with 10 AM MK571 or PSC833
were not significantly different (P = 0.34 and 0.4 ,
respectively) from those of eggs treated with the same
inhibitors (Figs. 4A, B). This indicates that the levels of c-
am hydrolysis by esterases and passive permeability are
equivalent in eggs and embryos; rather, fertilization results
in a change in transport-inhibitor sensitive ac tivity. Thu s, the
change in c-am accumulation that occurs post-fert ilization is
related to upregulation of transport activity. The results also
demonstrate that there is a low level of MK571-sensitive
transport in eggs prior to fertilization.
Fig. 5 shows continuous, time-lapse recording of calcein
accumulation in seven sea urchin eggs at fertilization. In this
experiment eggs were attached to a poly-lysine coated dish
and recordings were started immediately after addition of c-
am. Fluorescence was measured every 2 min and points
represent the mean fluorescence intensity in seven embryos
(Fone standard deviation). As seen, fluorescence increases
in a linear fashion prior to fertilization, indicating low efflux
activity. Within 20–30 min after fertilization, a leveling of
intracellular fluorescence accumulation occurs. This indi-
cates that a distinct increase in efflux activity begins at 20–30
min after fertilization, and a corresponding reduction in the
accumulation of intra cellular calcein is observed. The
addition of the mrp-specific inhibitor MK571 (10 AM)
post-fertilization restores the unfertilized rate of calcein
accumulation.
Mrp- and p-gp-specific embryo transport
The results in Figs. 3 and 4 show that both mrp and p-gp-
like transport activities are upregulated in embryos but
suggest that mrp is the maj or trans porter since the increase
in fluorescence is greater with the mrp-specific inhibitor
MK571 tha n with the p-gp inhibitors cyclosporin-A,
reversin 205, verapamil, and PSC833.
The results in Fig. 6 examine in greater detail the relative
contribution of p-gp and mrp transporters to c-am efflux in
embryos using the mrp- and p-gp-specific inhibitors,
MK571 and PSC833. PSC833 is e ffective at lower
concentrations but the effect saturates at about 3 AM and
increased levels of drug do not result in any additional
Fig. 2. Typical plot of calcein accumulation in unfertilized eggs and
embryos (250 nM extracellular c-am). Calcein-am is excluded from
embryos but in eggs it enters the cell, is hydrolyzed to fluorescent calcein,
and accumulates in a linear fashion over time (points represent mean F 1
SD of 20–100 individual embryos from four females).
Fig. 3. Change in intracellular fluorescence in eggs and 2-cell embryos treated with mdr inhibitors for 90 min, as compared to untreated controls of the same
developmental stage (bars represent means F 1 SD of 3 batches of eggs or embryos). There is a small increase in fluorescence intensity in eggs treated with the
mrp-specific inhibitor MK571 as compared to controls, but in embryos the fold change is much greater due to the significant decrease in calcein accumulation
that occurs at fertilization. MK571, 10 AM, is more potent than any of the other p-gp-specific or general mdr inhibitors at inhibiting transport in the embryos.
A.M. Hamdoun et al. / Developmental Biology 276 (2004) 452–462456
Page 5
accumulation. The effect of MK571 also saturates, but at a
higher concentration. Importantly, the amount of intra-
cellular fluorescence in saturating doses of MK571 is four
to five times higher than when transport is maximally
inhibited by PSC833, indicating a greater contribution of
efflux transport activity from mrp transporters. These results
indicate that both p-gp and mrp transport activity are present
in these embryos, but that the p-gp contribution is minor
compared to the mrp contribution.
Mechanism of transport initiation at fertilization
Translation and transcription
To determine the significance of transcription and trans-
lation in the upregulation of efflux transport, eggs were
fertilized and appropriate inhibitors added after confirmation
of fertilization envelope elevation (5 min post-fertilization
(PF)). At 60 min PF the inhibitors were washed out and c-
am was added. Embryos were incubated for 90 additional
min and fluorescence intensities were measured at 150 min
PF (2-cell stage).
Initiation of transport is not affected by inhibitors of
transcription or translation, actinomycin and e metine,
respectively (Fig. 7). As demonstrated previously (Kiefer
et al., 1969; Wagenaar, 1983), embryos progress through
first cleavage normally when treated with actinomycin but
arrest prior to first cleavage with emetine treatment. The
lack of effect of actinomycin and emetine on transport
activity suggests that transcription and translation do not
mediate post-fertilization changes of trans porter activity. In
addition, we directly measured the levels of transcripts of
SpABCC1 and SpABCB1 in eggs and early embryos by
real-time PCR (Table 1). These results showed an insignifi-
cant decrease in transcript abundance at fertilization
followed by a later increase in transcript abundance 90–
120 min PF. The increase in transcript abundance occurs
later than the increase in transporter activity, confirming that
the large induction in transporter activity occurs prior to de
novo transcription of transporters. It is also important to
note that the levels of these transcripts appear to be quite
low (b1 copy/egg) as determined by comparison of C
T
values in eggs to those from plasmid standards (data not
shown).
Role of microfilaments in upregulation of transport
Earlier work has shown that transport systems for amino
acids and nucleosides are also activated after fertilization
and that this process is prevented by microfilament
inhibitors (Epel, 1972; Schneider, 1985; Swezey et al.,
1987). The stimulus-induced delivery of vesicles to the
plasma membrane is typically a microfilament-mediated
process requiring actin polymerization (Ikebuchi et al.,
2001; Kato et al., 2004; Wessel et al., 2002). We examined
the role of microfilaments in upregulation of transport using
the actin polymerization inhibitors cytochalasin-D (CD) and
latrunculin-A (Fentean y and Zhu, 2003; Schatten et al.,
1986; Wessel et al., 2002) and found that both compounds
prevented the upregulation of efflux transport activity since
significantly more calcein accumulated in the embryos
fertilized in these inhibitors than controls (Fig. 7). Because
Fig. 4. (A) Absolute fluorescence levels in eggs and embryos before and
after treatment with mrp- and p-gp-s pecific inhibitors (MK571 and
PSC833; bars represent means F 1 SD of 3 batches of eggs and embryos).
The addition of inhibitors to embryos restores fluorescence to similar
levels to eggs suggesting that bpassiveQ permeability of eggs and embryos
is similar but that MK571 and PSC833 sensitive transport increase at
fertilization. (B) Representative pseudo-color micrographs of eggs
embryos treated with mrp and p-gp inhibitors. Color bar (left) indicates
relative fluorescence intensity (white is brightest fluorescence, violet
dimmest).
Fig. 5. Time-based measurement of calcein accumulation in eggs (n =7)
before, during and after fertilization. Calcein accumulates in a linear fashion
in unfertilized eggs (A) but calcein accumulation stops within 25 min of
fertilization (B) and remains at a steady state (C) until the mrp-specific
inhibitor MK571(10 AM) is added (D). These results indicate that c-am
transport is activated at fertilization.
A.M. Hamdoun et al. / Developmental Biology 276 (2004) 452–462 457
Page 6
these inhibitors were added 5–10 min after fertilization and
washed out prior to c-am addition this effect was not due to
their competitive inhibition of transport activity.
Latrunculin caused a nearly 30-fold increase in calcein
accumulation and its efficacy as an irreversible microfila-
ment inhibitor was also evidenced by the fact that it blocked
cell division, even though it was washed out (to prevent any
possible interaction with the transporters as a competitive
substrate for efflux) at 60 min post-fertilization. In contrast,
with 2 AM cytoch alasin-D, half of the embryos compl eted
the first cell division consistent with reversible inhibition of
actin polymerization in these embryos (Wessel et al., 2002).
In cleavage-arrested, CD-treated embryos, there was a 7-
fold increase in fluorescence relative to controls, indicating
inhibition of the transporter. However, in CD-treated
embryos that divided, fluorescence levels were simil ar to
controls and we observed this effect even with 4 AMCD
(not shown) suggesting recovery from CD treatment, rather
than threshold dosage effects (Wessel et al., 2002). These
results show that upregulation of transporter activity at
fertilization is microfilament dependent.
Role of efflux transport in the embryo
Protection from xenobiotics
We tested the hypothe sis that the major, mrp-
mediated, transport activity protects embryos from
xenotoxins by me asuring the toxicity of the mrp
substrate vinblastine to embryos during the first cell
division in the presence or absence of 5 AM MK571. As
seen in Fig. 8, MK571 increases sensitivity to vinblastine
by at least one order of magnitude confirming that mrp
activity is protective.
Progression through the cell cycle
We also observed that treatment of embryos with 10 A M
or higher levels of MK571 alone, retards the first cell
division. To analyze this in depth, we fixed embryos at
various intervals following fertilization and chromosomal
events during the cell cycle were visualized using the
fluorescent DNA probe Sytox green. We found that
embryos treated with 10 AM MK571 take a longer time to
complete anaphase than do controls, though progression
through earlier stages was not affected (Fig. 9A). Although
the 10 AM MK571-treated embryos eventually complete cell
division and continue to divide, many develop abnormally
and have unevenly sized blast omeres (Fig. 9B) As opposed
to the 10 AM concentration of MK571, concentrations of 15
AM or higher slowed progression through all stages of the
cell cycle, and concentrations in excess of 20 AM stop cell
division all together (not shown).
Discussion
This study shows a post-fertilization upregulation of
efflux transporter activity that begins 25 min post-fertiliza-
tion, coincident with the late r responses to fertilization that
include delivery of other stored maternal proteins to
embryonic surfaces (Matese et al., 1997), pronuclear fusion,
and DNA synthesis (Epel, 1990). The confluence of
changes at this time may indicate a second phase of the
post-fertilization events that is linked to resumption of the
cell cycle (Jaffe et al., 2001).
This efflux activity is sensitive to inhibitors of mdr
transporters, including specific inhibitors of p-gp and mrp,
PSC833 and MK571, respectively ( Gekeler et al., 1995; Liu
et al., 2001; Mayer et al., 1997; Miller et al., 2002a), and is
most effectively blocked by the mrp-specific inhibitor
MK571. The data indicate that mrp-like activity is the
major high-capacity transport activity in the sea urchin egg
and embryo but the results also reveal a lower capacity, p-
gp-like transport. The mrp activity appears to have several
Fig. 6. Representative dose–response curves of the p-gp- and mrp-specific
inhibitors (PSC833 and MK571, respectively) on calcein-am transport in 2
cell embryos. PSC833 inhibits transport at low doses, but MK571 is much
more effective at high doses.
Fig. 7. Transcription and translation (actinomycin-D and emetine) inhibitors
do not block the initiation of transport at fertilization, but cytoskeleton
disrupting compounds cytochalasin-D and latrunculin-A do. This suggests
that the initiation of transport occurs in some microfilament-dependent
process such as movement of vesicles to the plasma membrane.
A.M. Hamdoun et al. / Developmental Biology 276 (2004) 452–462458
Page 7
roles. Inhibition of activity enhances toxicity of vinblastine,
indicating that this efflux transport can protect against
xenobiotics, as seen in other organisms. Inhibition of the
transporter also retards progressio n through the cell cycle,
with major effects on anaphase. This effect on mitosis and
the timing of upregulation of activity, point to a role for
efflux transport in promoting entry or progression through
the first cell division.
Our molecular data demon strate that several mdr trans -
porter mRNAs are present in the egg including SpABCB1,
SpABCC1, and SpABCC2, and that they encode gene
products that could play roles in the observed xenobiotic
efflux. SpABCB1 encodes an efflux trans porter similar to p-
gp, an extensively studied xenobiotic transporter that
transports c-am and various xenobiotics in mammalian cells
(Ambudkar et al., 1999). SpABCC1 encodes a protein with
homology to mammalian mrp5 which is an efflux trans-
porter with broad substrate specificity and the capacity to
transport characteristic mrp substrates including cyclic
nucleotides, anti-viral drugs and anti-cancer drugs (Kruh
and Belinsky, 2003). SpABCC1 was highly represented in
the sea urchin egg cDNA library and an analysis of the
translated partial cDNA shows that the protein it encodes
shares 65% amino acid identity to murine mrp5 in the
conserved ABC transporter ATPase domain and an overall
identity of about 22% to the same murine protein. In plants
mrp5 homologs establish auxin gradients that regulate root
development (Friml et al., 2003; Gaedeke et al., 2 001).
SpABCC2 encodes a protein with homology to mammalian
sulfonylurea receptors which regulate K+ conductance in
insulin-sensitive cells and with transport properties similar
to those of mrp5 (Bryan and Aguilar-Bryan, 1997; Kruh and
Belinsky, 2003).
In addition to molecular approaches, different fluoro-
phores are used to identify the transporters responsible for
particular transport phenotypes (see Litman et al., 2001 for
review). For example, while c-am is extruded by both p-gp
and mrp proteins, rhodamine is only transported effectively
by cells that express p-gps. Consistent with this observation,
Urechis embryos and larvae express p-gp, and are capable
of high-capacity rhodamine transport, while sea urchin eggs,
embryos and larvae are not (Hamdoun et al., 2002; Toomey
and Epe l, 1993). It is probable that these same studies did
not identify drug-sensitive p-gp transport in sea urchin eggs
and embryos because rhodamine was used at concent rations
(1 AM) that saturate the low-capacity p-gp-like transport we
identified here.
Similarly, our previous studies did not detect p-gp
protein (Hamdoun et al., 2002; Toomey and Epel, 1993)
on western blots of sea urchin egg proteins using the
monoclonal antibody C219 which reacts strongly with the
conserved the epitope VQEALD (Georges et al., 1990). The
corresponding epitope in sea urchin p-gp has a single amino
acid substitution of the glutamate with aspartate, which
could account for the lack of immunoreactivity of C219
with sea urchin p-gp. Given the molecular evidence and
PSC833 sensitivity, it is clear that sea urchin embryos have
some p-gp-mediated efflux activity.
Analysis by real-time-PCR (RT-PCR) shows that
SpABCC1 and SpABCB1 are rare transcripts and that
their level does not significantly increase until 90–120 min
post-fertilization. In contr ast, transport activity increases
within 25 min of fertilization and the increase in efflux
acti vity occurs in the prese nce of protein and RNA
synthesis inhibitors. These data then all indicate that the
Table 1
Results of real-time q-pcr analysis of mrp (SpABCC1) and p-gp (SpABCB1) transporter mRNA abundance before and after fertilization are shown
Time PF
(min)
C
T
18s rRNA C
T
SpABCC1 C
T
SpABCB1 Fold change SpABCC1
(relative to egg)
Fold change SpABCB1
(relative to egg)
0 14.3 (F0.4) 34.4 (F1.1) 29.1 (F0.8)
30 15.8 (F1.2) 37.4 (F1.6) 31.4 (F2.8) 5(F3.4) 2(F3.2)
60 16.6 (F0.4) 37.2 (F0.9) 31.7 (F1.5) 0.37 (F1.9) 0.25 (F0.4)
90 15.6 (F1.5) 35.9 (F0.4) 30.7 (F2.6) 0.9 (F1.8) 0.2 (F0.9)
120 15.0 (F0.5) 34.5 (F2.0) 28.9 (F0.8) 4 (F3.2) 2.4 (F1.5)
The cycles to threshold (C
T
) values for each transcript measured are shown. The high C
T
values for mrp and p-gp suggest that these are low abundance
transcripts. A standard curve relative to a recombinant standard suggests they are both at b1 copy/egg (not shown). The fold change in mrp and p-gp is
expressed relative to the value of the internal standard (ribosomal RNA) for each sample. Given the low abundance of these transcripts, the changes in
transcript abundance are not likely to be significant. Values represent the means (Fone standard deviation) for three batches of eggs and embryos.
Fig. 8. Competitive inhibition of mrp activity with 5 AM MK571 sensitizes
the embryo to vinblastine measured as failure to enter and complete first
cell division. Vinblastine was added 45 min PF (after efflux transport is
initiated) either in the presence or absence of MK571 (n = 100 embryos per
point). At 145 min PF embryos were fixed and scored for completion of
first cell division.
A.M. Hamdoun et al. / Developmental Biology 276 (2004) 452–462 459
Page 8
upregulation of mdr efflux activity at fertilization is
mediated post-translationally.
The upregulation of mrp following fertilization may
share a common mechanism with the previously described
increase in activity of amino acid transporters after
fertilization. Activation of mrp and amino acid transport
activity are both insensitive to pharmacological inhibition of
transcription or translation, sensitive to microfilament
polymerization inhibitors and activity becomes apparent
after cortical exocytosis (Epel, 1972; Schneider, 1985). This
similar behavior among transporters suggests that they are
translocated to the plasma membrane in vesicles after
fertilization. Indeed, this may be part of a cascade of vesicle
movement events after fertilization which also includes
movement and exocytotic deposition of vesicle contents
(Matese et al., 1997) and, in at least some cases, this
movement is similarly carried out on microfilaments (Kato
et al., 2004).
This phenomenon is analogous to numerous other types
of signal-induced changes in cellular transport physiology
that are mediated by delivery of transporters to the plasma
membrane (see Bryant et al., 2002 for review), including the
well-studied example of insulin-induced translocation of the
glucose transporter GLUT4 to the membrane (Watson and
Pessin, 2001). The microfilament-mediated increase in mrp
activity in the sea urchin embryo at fertilization may also be
accompanied by other post-translational events such as
protein kinase-mediated upregulation of pre-existent and/or
newly inserted membrane transporter activity (Miller et al.,
2002b).
One role of these transporters at fertilization is protec-
tion, as is demonstrated by the sensitization of embryos to
vinblastine by competitive inhibition of mrp activity with
MK571. This role is similar to the protective functions
ascribed to these transporters in somatic cells and consistent
with their expression in the adult blood–brain barrier (Miller
et al., 2002a), blood–testis barrier (Melaine et al., 2002), and
in intestinal epithelia (Miller et al., 2002b). Similarly, this is
also consistent with a role for these transporters in
protection of embryos and fits with their expression both
in embryos (Broeks et al., 1996; Elbling et al., 1993;
Toomey and Epe l, 1993) and the placenta (Leazer and
Lassen, 2003; Pascolo et al., 2003).
An unexpected finding was that embr yos treated with
MK571 alone exhibit a mitotic delay seen as a retardation
of progression through anaphase at 10 AM MK571 and
overall retardation at higher levels of the inhibitor. One
possibility is that the effect is a non-specific inhibit ion of
one of the enzymes involved in mitotic progression, such
as polo or aurora kinase. A second possibility is that the
transporter is pumping out some generally toxic substance
produced by metabolic activity and when this is not
removed cell activity is retarded with anaphase being
particularly sensitive. A reasonable candidate in this
regard is 4-hydroxynonenal which is a toxic lipid
peroxidation product associated with free radical produc-
tion and which is a known mrp substrate (Renes et al.,
2000). A third possibility is that there is some regulatory
substance that needs to be removed in order for the cell to
enter anaphase. Regulatory proteins are released by the
kinetochore and most emphasis has focused on inductive
roles o f these agents; perhaps, there is also a negative
Fig. 9. (A) Graph shows the progression of control and 10 AM MK571-
treated (5 min PF) embryos through first cell division (n = 50–100
embryos per point). Progression through metaphase is similar in controls
and MK571-treated embryos. In contrast at 90 min post fertilization N60%
of control embryos are past anaphase, while virtually none of the MK571-
treated embryos have completed this sta ge. These MK571-treated
embryos later complete anaphase demonstrating MK571-induced devel-
opmental delay rather than cell death. (B) Micrograph. (i) Control
embryos enter prophase of the second cell division by 120 min following
fertilization. In contrast, cell division is retarded in MK571-treated
embryos and anaphase is especially prolonged (ii). Embryos treated with
10 AM MK571 eventually complete first cell division but some of them
develop abnormalities, including uneven blastomeres, as seen in the 32-
cell embryos shown (iii and iv).
A.M. Hamdoun et al. / Developmental Biology 276 (2004) 452–462460
Page 9
agent that must be effluxed from the cell for anaphase to
commence.
Possible candidates for such a negative agent in sea
urchins are leukotrienes, which are derived from arach-
idonic acid oxidation by specific lipoxygenases and which
are mrp substrates (see Funk, 2001 for revie w). Indeed, the
active moiety of MK571 is a leukotriene analog and
MK571 can also act as a leukotriene receptor antagonist,
but the K
d
for the receptor antagonism is in the 10 nM range
(Lynch et al., 1999), whereas transport inhibition occurs in
the 10-AM range which is also where the effect on mitosis is
observed. Leukotrienes have signal transduction roles and
most work has focused on them as paracrine effectors
(Funk, 2001).
If leukotrienes are involved in regulating mitosis in the
one cell stage of the sea urchin embryo they most likely
act intracellular ly, perhaps as some part of a checkpoint,
and efflux from the cell then allows progression through
the cell cycle. Silver has noted changes in leukotrie nes
during the cell cycle in sand dollar eggs and posited an
effect on nuclear envelope breakdown (Silver, 2001). Perry
and Epel (1985) and Hawkins and Brash (1997) have
described lipoxygenase activities in sea urchin eggs which
similarly point to active arachidonic acid metabolism in
these cells. However, a specific 5-lipoxygenase has not
been identified.
This study points to dual roles for mrp transporters in
development. One is the well-known effect on toxicant
efflux. The other i s a possible role i n pumping ou t
endogenous compounds as part of cell regulation. Such
roles have recently been described for establishing auxin
gradients in Arabidopsis (Friml et al., 2003) and differ-
entiation in Dictyostelium ( Good and Kuspa, 2000).
Acknowledgments
We thank Drs. Kathy Foltz and Forest O’Neil for their
generous assistance with screening of cDNA macroarray
libraries. Drs. Julie Alipaz and Stephen Palumbi are
thanked for providing expert guidance with sequencing
of the transporter genes and assembly of the sequences.
Ms. Nature McGinn is thanked for her assistance with
embryo imaging. Dr. Christian Leutenegger provided
technical support for q-pcr. This work was funded in part
by: UC Coastal Toxicology, Research and Teaching
Program (GC), by a NICHD Training Grant (AH) T32
HD071131, bFertilization and Early DevelopmentQ and by
the National Sea Grant Program, National Oceanic and
Atmospheric Administration, US Department of Commerce
under project number R/CZ-182 through the California Sea
Grant College (DE). The views expressed herein are those
of the authors and do not necessarily reflect the views of
NOAA or a ny of its subagencies. The US government is
authorized to reproduce and distribute for governm ental
purposes. BML contribution number 2217.
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    • "The course of the concentration-effect curves included a clear maximum, confirming that concentrations of maximal effects were indeed comprised in the concentration series for all three inhibitor compounds. Likewise, differing effect kinetics of ABCC and ABCB1 inhibitors were found in dye accumulation assays with D. polymorpha embryos and larvae (Navarro et al., 2012) and with native cells or tissues from other aquatic invertebrates (Hamdoun et al., 2004;). Specificity of inhibition of ABCB1-and ABCC1-type efflux activities in native bivalve tissues by certain inhibitory compounds was indicated earlier when it was shown that the combination of the two inhibitors PSC833 and MK571 resulted in a larger effect amplitude of calcein accumulation in tissue than with single inhibitor compounds. "
    [Show abstract] [Hide abstract] ABSTRACT: Aquatic organisms, such as bivalves, employ ATP binding cassette (ABC) transporters for efflux of potentially toxic chemicals. Anthropogenic water contaminants can, as chemosensitizers, disrupt efflux transporter function enabling other, putatively toxic compounds to enter the organism. Applying rapid amplification of cDNA ends (RACE) PCR we identified complete cDNAs encoding ABCB1- and ABCC1-type transporter homologs from zebra mussel providing the molecular basis for expression of both transporter types in zebra mussel gills. Further, efflux activities of both transporter types in gills were indicated with dye accumulation assays where efflux of the dye calcein-am was sensitive to both ABCB1- (reversin 205, verapamil) and ABCC1- (MK571) type specific inhibitors. The assumption that different inhibitors targeted different efflux pump types was confirmed when comparing measured effects of binary inhibitor compound mixtures in dye accumulation assays with predictions from mixture effect models. Effects by the MK571/reversin 205 mixture corresponded better with independent action, whereas reversin 205/verapamil joint effects were better predicted by the concentration addition model indicating different and equal targets, respectively. The binary mixture approach was further applied to identify the efflux pump type targeted by environmentally relevant chemosensitizing compounds. Pentachlorophenol and musk ketone, which were selected after a pre-screen of twelve compounds that previously had been identified as chemosensitizers, showed mixture effects that corresponded better with concentration addition when combined with reversine 205 but with independent action predictions when combined with MK571 indicating targeting of an ABCB1-type efflux pump by these compounds.
    Full-text · Article · Mar 2016 · Toxicology and Applied Pharmacology
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    • "Surviving individuals were then maintained as clonal lineages for several generations under non-contaminated media and then their acute tolerance assayed following conventional procedures (OECD, 1997). Selected chemicals included the cytostatic drugs that are substrates of mammalian ABCG2, ABCB1 and ABCC1 (mitoxantrone) and ABCC1 transporters (chlorambucil) (Litman et al., 2000; Morrow et al., 2006; Su et al., 1998) and are toxic to D. magna (Campos et al., 2014), verapamil, which is a known ABCB1 inhibitor in mammals, mussels and sea urchins (Eufemia and Epel, 2000; Faria et al., 2011; Hamdoun et al., 2004; Yusa and Tsuruo, 1989); pentachlorophenol, which is a known inducer of Abcb1 transporter activity in mussels and D. magna (Campos et al., 2014; Eufemia and Epel, 2000 ) and ivermectin , which is an antihelmintic drug known to be a substrate of MXR transporter (Alvarez et al., 2006). "
    [Show abstract] [Hide abstract] ABSTRACT: Multixenobiotic resistance mechanisms (MXR) were recently identified in Daphnia magna. Previous results characterized gene transcripts of genes encoding and efflux activities of four putative ABCB1 and ABCC transporters that were chemically induced but showed low specificity against model transporter substrates and inhibitors, thus preventing us from distinguishing between activities of different efflux transporter types. In this study we report on the specificity of induction of ABC transporters and of the stress protein hsp70 in clones selected to be genetically resistant to ABCB1 chemical substrates. Clones resistant to mitoxantrone, ivermectin and pentachlorophenol showed distinctive transcriptional responses of transporter protein coding genes and of putative transporter dye activities. Expression of hsp70 proteins also varied across resistant clones. Clones resistant to mitoxantrone and pentachlorophenol showed high constitutive levels of hsp70. Transcriptional levels of the abcb1 gene transporter and of putative dye transporter activity were also induced to a greater extent in the pentachlorophenol resistant clone. Observed higher dye transporter activities in individuals from clones resistant to mitoxantrone and ivermectin were unrelated with transcriptional levels of the studied four abcc and abcb1 transporter genes. These findings suggest that Abcb1 induction in D. magna may be a part of a general cellular stress response.
    Full-text · Article · Mar 2016 · Aquatic toxicology (Amsterdam, Netherlands)
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    • "This reduces the tests' sensitivity to chemosensitisers and affects their comparability to in and ex vivo systems. Ex vivo tests rely on dye or radiolabeled marker accumulation, i.e., in mussel gill tissue, sea urchin larvae and mussel early life stages (Cornwall et al., 1995; Hamdoun et al., 2004; McFadzen et al., 2000). Unfortunately, it is not possible to systematically determine the extent to which in vitro and in/ex vivo test results differ quantitatively , as a reliable pair of ICs is only available for verapamil (Table SI2.1, SI). "
    [Show abstract] [Hide abstract] ABSTRACT: The active cellular efflux of toxicants is an efficient biological defense mode present in all organisms. By blocking this so-called multixenobiotic resistance transport-a process also referred to as chemosensitisation-, cellular bioaccumulation and the sensitivity of organisms towards environmental pollutants can increase. So far, a wide range of compounds, including pesticides, pharmaceuticals, fragrances, and surfactants, have been identified as chemosensitisers. Although, significant on a cellular level, the environmental impact of chemosensitisation on the organism level is not yet understood. Critically evaluating existing data, this paper identifies research needs to support our tentative conclusion that chemosensitisation may well enhance the risks of chemical exposure to aquatic organisms. Our conclusion is based on studies investigating the impact of individual chemicals and complex environmental mixtures on aquatic wildlife and a chemosensitiser mixture toxicity model which, however, is subject to great uncertainty due to substantial knowledge gaps. Those uncertainties include the inconsistent reporting of effect data, the lack of representative environmental contaminants tested for chemosensitisation, and the publishing of highly unreliable nominal exposure concentrations. In order to confirm the tentative conclusion of this paper, we require the significant and systematic investigation of a broader set of chemicals and environmental samples with a harmonised set of bioassays and rigorously controlled freely dissolved effect concentrations. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Jul 2015 · Aquatic Toxicology
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