ABSTRACT
The echluroid worm Urechis caupo is one of the many marine
invertebrates that is able to live and reproduce in a polluted
environment. One proposed explanation for this worm's resistance
to pollutants is the multixenobiotic resistance (MXR) mechanism,
whereby a protein in the cell membrane pumps a wide variety of
moderately hydrophobic compounds out of the cells. This protein is
analogous to multidrug resistance (MDR) in mammalian cells which
provides mammalian cells with multidrug resistance to
chemotherapeutic agents. These data support the role of MXR as a
protective mechanism for marine invertebrates, suggest bioassays
for pollutants, and propose that the phenomenons of hormesis and
multixenobiotic resistance are related.
INTRODUCTION
Multidrug resistance, or multixenobiotic resistance, is one
phenomenon that is responsible for both the resistance of tumor
cells to chemotherapy drugs, and the ability of marine organisms to
survive in highly polluted habitats. This resistance is believed to be
directly related to levels of expression of the MDR protein in
mammals, and to an immunologically related protein of 140-145 KD
in marine invertebrates (1). For a protein with such diverse effects
as are created by MDR, one can easily imagine the importance of
scientific research of MXR to the fields of medicine, toxicology, and
marine biology.
The MDR protein is a transmembrane protein of 1280 amino
acids with two equivalent ATP binding sites, only one of which must
be occupied for transport (2). A protein immunologically related to
the MDR protein in mammals has been shown to exist in the
echiuroid worm Urechis caupo (3) which lives and reproduces in U¬
shaped burrows in the mud flats at Moss Landing. Urechis uses
peristaltic contractions of its muscular body to move water through
the burrows, thereby replenishing food and oxygen, and removing
wastes that have seeped in from the surrounding sediment. Previous
studies have shown that hydrogen sulfide, a highly toxic compound,
can also accumulate in sediments of shallow marine bays such as the
Elkhorn Slough at Moss Landing (4).
In this study I assessed MR activity using rhodamine
fluorescence assays which measure the accumulation of rhodamine
dye, a substrate of the mammalian multidrug transporter (5).
Urechis embryos incubated with rhodamine and a competitive
inhibitor of the MXR protein fluoresce more brightly than embryos
in the dye alone. Verapamil is a potent inhibitor of the MDR protein
(6,7) and it was used as a known inhibitor in the rhodamine assays.
The second part of this study asked whether MDR activity can
protect embryos that develop in supposedly polluted sediment water
or in the presence of known cytotoxic compounds. For this study I
devised an assay in which Urechis caupo embryos or
Strongylocentrotus purpuratus embryos are allowed to develop in
sediment pore water in the presence, or absence of an MXR
inhibitor. If compounds that are substrates for the MXR protein are
present in the pore water, competition between those substrates and
the NXR inhibitor would result in higher levels of substrates inside
the cells and, possibly, abnormal development.
Through the use of these two methods, I investigated the
presence of substrates for the MXR protein in sediment from Moss
Landing. In addition the effectiveness of various chemicals as
competitive inhibitors of MXR in the developmental assay, and the
effects of sediment on the development of Urechis caupo and
Strongylocentrotus purpuratus embryos were determined.
The results of this work revealed the phenomenon of
hormesis, in which a stress response is stimulated in an organism
that is exposed to non-toxic levels of a toxin (8). I also found
evidence which suggests that verapamil is toxic at very low levels
(.022 uM), and that this compound would give misleading results
when used as a competitive substrate in developmental assays. On
the other hand, my work suggests that rhodamine B might be a
useful competitive substrate in developmental assays, because it is a
known substrate of the MXR protein and it is less toxic than
verapamil.
MATERIALS AND METHODS
Adult Urechis caupo were collected at the mud flats in Moss
Landing, CA on two different dates, April 9, 1993 or May 28, 1993.
The worms were maintained in tanks with a constant source of fresh
sea water. Urechis were spawned, and the eggs were fertilized
according to Gould (9). Strongylocentrotus purpuratus were
collected at Point Arena, CA, and they were maintained with a
constant flow of fresh sea water according to Czihak and Peter (10).
For the rhodamine fluorescence assays 10 ul of 1mM
rhodamine B was added to 10 ml of eggs, which made a final
concentration of luM. The eggs were divided into 2 test tubes of 5
ml each, and either 22pM of verapamil or 25 ul of sediment extract
was added to one of the test tubes. The embryos were incubated for
one hour at 16’C. The eggs were then washed into filtered sea
water and the fluorescence was measured using a Zeiss
epifluorescence microscope fitted with a photosensor. The light
emitted by the embryos was expressed by a voltmeter as relative
fluorescence units.
For the developmental assays the eggs were fertilized in type
GS.22um millipore filtered sea water (MFSW) and incubated in their
respective solutions at 16’C. These solutions were combinations of
the following: MFSW, pore water, verapamil (.022 uM or .044 uM),
rhodamine B (1.25 uM), and emetine (.0025 uM or .005 uM). In
cases where pore water was used, it was used in equal amounts with
the MFSW. Pore water was removed from the sediment by
vigorously shaking it for two minutes, and centrifuging it at high
speed for one minute. To reduce water loss by evaporation, the
beakers containing the Urechis embryos were partially covered with
Parafilm, whereas the S. purpuratus embryos were constantly stirred
to prevent crowding and anoxia. At the designated times an aliquot
of embryos was removed, inspected for abnormalities under the
microscope, counted and discarded. If the embryos had hatched
they were fixed with one drop of 37% formaldehyde before counting
and inspection.
Abnormal development for sea urchin embryos included
delayed cleavage, lysed cells, disorganized cells, exogastrulation,
and little or no movement compared to controls. In the
developmental assay for Urechis, abnormal development was based
on the same critéria, except that once the embryos were swimming,
continuous spinning to only one side was considered abnormal, in
addition to little movement compared to controls.
Sediment samples were collected at Moss Landing, at the site
of adult worm collection. They were frozen at -80 C within one
hour of collection, and thawed prior to use. For some experiments
extractions from the pore water, or directly from the sediment were
done by mixing with chloroform:methanol (2:1). The solvent phase
was removed to a clean tube and evaporated. The remaining residue
was dissolved into 200 ul of 95% ethanol and used in the rhodamine
or development assays.
RESULTS
Rhodamine Assays:
The embryos incubated with 1 uM rhodamine plus 22uM
verapamil have average fluorescence levels that are 15.6% higher
than those embryos incubated in only MFSW and rhodamine. This
percentage is a measure of the effect of verapamil on MXR activity.
The embryos incubated in luM rhodamine plus 25 ul of sediment
extract fluoresced an average of 11.65% more than the embryos
that were incubated in only MFSW plus rhodamine. This indicates
that there are substrates for MDR in the sediment, but that they are
not in as high a concentration as the 22uM verapamil substrate.
One should also note that the intensity of fluorescence for the
sediment extract varied from 5.5% to 17.8%, indicating that the
levels of substrates in the sediment are not constant.
S. purpuratus Developmental Assays:
The embryos incubated in pore water, or water taken from the
sediment after centrifugation, averaged 51.8% more abnormalities
than the embryos that developed in MFSW. It is believed that this
high level of abnormalities in the pore water can be attributed to the
combinatory effect of toxins in the pore water, and the lack of an
MXR protein to protect the sea urchin embryos. Less clear results
were found in the developmental assay done with a sediment
extract. The sediment extract was made by evaporating with
chloroform: methanol (2:1), and then dissolving the residue in
ethanol. Although the embryos that were incubated in sediment
extract averaged 59.7% higher abnormalities than those that
developed in only MFSW, the control embryos that were incubated
in an equal amount of ethanol also had very high abnormality,
which indicates that the ethanol was affecting development. The
numbers for S. purpuratus and Urechis developmental assays were
calculated by taking the average number of abnormalities in each
group, subtracting the difference between the two groups being
compared, and multiplying that number by 100 to get the %
abnormality.
U. caupo Developmental Assays with Verapamil 022 UM
Or.044 UM):
The first assay done with .022 uM of verapamil showed a
difference of only .66% between the average abnormality level of
the embryos developing in pore water plus verapamil and those
embryos developing in MFSW plus verapamil. However, when the
concentration of verapamil was doubled to .044 uM the difference
in abnormality levels increased to 18%. It is important to note that
after the sixth cleavage the controls of only verapamil increase in
abnormalities until they have a higher % abnormal than the
experimentals. One should also note that the % abnormal for the
embryos in the MFSW is higher than it is for the embryos developing
in only pore water. This result led to the idea that the phenomenon
of hormesis is related to the activity of MXR.
U. caupo Developmental Assays with Emetine (0025 uM or
005 UM):
The embryos that developed in pore water plus .0025 uM of
emetine averaged only 3.6% more abnormalities than did the
embryos that developed in MFSW plus an equal concentration of
emetine. However, when the concentration of emetine was doubled,
those embryos that were incubated in pore water plus emetine
averaged 19% more abnormalities than did those embryos that were
incubated in only MFSW plus emetine. As in the developmental
assay with verapamil, the levels of abnormalities for all the embryos
increased after the sixth cleavage. More importantly, by the sixth
cleavage the % abnormal for the embryos in the MFSW was higher
than it was for those developing in only pore water, which provides
further evidence for hormesis.
U. caupo Developmental Assay with Rhodamine B (1.25
UM:
The embryos that developed in pore water plus 1.25 uM of
rhodamine averaged 8.8% more abnormalities than those embryos
that developed in MFSW plus the same concentration of rhodamine.
As in the previous assays with Urechis, those embryos that
developed in only MFSW eventually averaged a higher % abnormality
10
than did those embryos that developed in only pore water.
Although the abnormalities of the control group increased after the
sixth cleavage, rhodamine proved overall to be much less toxic than
either verapamil or emetine.
11
DISCUSSION
In this study and previous studies (3), it has been shown that
the efflux of rhodamine B dye is decreased in the presence of pore
water or extractions of sediment collected from the mud flats at
Moss Landing. Based on rhodamine fluorescence assays that I have
done, which reveal varying degrees of rhodamine efflux inhibition, it
is possible to conclude that although there is indirect evidence of
MXR substrates in the sediment, the levels of these substrates vary.
These different levels may be depend on the time of day or of year
that the sediment is collected, the location of the sediment, the
biological make-up of the sediment and/or the size of the sediment
grains.
The developmental assays done with Strongylocentrotus
purpuratus provide evidence that toxic substrates for the MXR
protein exist in the sediment collected from Moss Landing. S.
purpuratus has been shown to lack the MXR protein (3), and this
species of sea urchin develops abnormally in the sediment which
Urechis caupo inhabits. Therefore, it is reasonable to propose that
the abnormal development in S. purpuratus is due to the absence of
MXR in this species of sea urchin, and that MXR is a protective
mechanism for inhabitants of the Moss Landing mud flats.
The developmental assays done with Urechis demonstrate that
the use of verapamil as a competitive substrate should be avoided
due to its toxic effects at concentrations as low as .022 uM.
Furthermore, it seems that rhodamine B may be used instead of
verapamil as a competitive inhibitor of MXR, because it is less toxic
and it is a known substrate of the MXR protein. These assays could
potentially become important methods of environmental xenobiotic¬
risk assessment and toxicology; especially in the case where the
effect of a specific toxin on development is known and can be
detected in embryos developing under the influence of a known
competitive inhibitor of the MXR protein.
Perhaps the most interesting and novel results in this project
are those that provide evidence for the phenomenon of hormesis
occurring in developing Urechis embryos. Hormesis is the condition
where low levels of a toxin stimulate a stress response in an
organism which instills, in that organism, some form of beneficial
effect(8). The lower numbers of abnormal embryos developing in
pore water than those developing in MFSW are evidence of hormesis.
However, further study is necessary to determine if the form of
hormesis that is occurring involves induction or activation of higher
levels of the MXR protein. If the levels of toxins in the environment
of Urechis caupo are affecting the expression or activation of the
MXR protein, then measurements of this protein may one day be
used as biomarkers of water pollution.
13
ACKNOWLEDGEMENTS
I would like to thank my advisor Dr. David Epel for sharing his
wonderful enthusiasm for development, and his valuable insights
into my project. Sincere thanks also go to Barbara Holland Toomey,
for taking the time and patience to share her knowledge of
multixenobiotic resistance in Urechis caupo, and the methods for
studying this phenomenon. Thanks to Chris Patton for his technical
assistance, as well as his "pre-med munchkin" jokes. Finally, I would
like to thank my colleague Joyce Chen, whose wit and
encouragement made an adventure out of the long days in lab.
LITERATURE CITED
1. Kurelec, B., Critical Reviews in Toxicology 22(1), 23-43 (1992).
2. Gottesman M. M., and Pastan, I., J. Biol. Chem. 263, 12163-
12166 (1988).
3. Toomey, B. H., and Epel, D., (submitted). Multixenobiotic
transport in Urechis embryos: protection from environmental
toxins.
4. Julian, D. and Arp, A. J., J. Comp. Physiol. B. 162, 59-67 (1992).
5. Mierendorf, R. C., C. Percy, and R. A. Young, in Methods in
Enzymology, S. L. Berger and A. R. Kimmel, Eds. (Academic Press,
Inc., Orlando, 1987) pp. 458-469.
6. Cornwell, M. M., Pastan, I. and Gottesman, M. M., J. Biol. Chem.
262, 2166, (1987).
7. Hollt, V., Kouba, M., Dietel, M. and Vogt, G., Biochem. Pharmacol.
43, 2601, (1992).
8. Dictionary of Scientific and Technical Terms, (McGraw-Hill Book
Company, New York, 1978), p. 764.
9. Gould, M., Methods in Developmental Biology, Wilt, F. H. and
Wessells, N. K., Eds. (Thomas Y. Crowell Co. New York, 1967) pp.
163-171.
10. Czihak, G. and Peter, R., Eds. The Sea Urchin Embryo, (Springer¬
Verlag, New York, 1975) pp. 11-23.
11. Newby, W., Memoirs of the American Philosophical Society, vol.
16. Independence Square, Philadelphia, Pa., (1940).
15
FIGURE LEGENDS
Figure 1. Rhodamine fluorescence vs. incubation solution. The first
column represents eggs that were incubated in only rhodamine dye,
and the second column represents eggs that were incubated with
rhodamine dye plus verapamil for the same amount of time. The
third column is the same as the first, just rhodamine dye incubation,
and the last one represents an incubation of rhodamine dye and
sediment extract.
Figure 2. Rhodamine fluorescence vs. incubation solution. The first
column represents eggs that were incubated in only rhodamine dye,
and the second column represents eggs that were incubated with
rhodamine dye plus verapamil for the same amount of time. The
third column is the same as the first, just rhodamine dye incubation,
and the last one represents an incubation of rhodamine dye plus
sediment extract.
Figure 3. Examples of normal and abnormal development in S.
purpuratus. The photographs were taken at the 16 cell stage of
embryos from the same batch of eggs. The normal embryo was
developing in MFSW, and the abnormal embryo, which exhibits
delayed cell cleavage, was developing in pore water.
Figure 4. Developmental assay with % abnormal plotted against time
in hours after fertilization. There is little difference between the
lines for the sediment extract and the ethanol, indicating that the
ethanol was affecting development.
Figure 5. Examples of normal and abnormal development in Urechis
caupo embryos. The photographs were taken at the swimming
trocophore stage of embryos from the same batch of eggs. The
16
normal embryo was developing in MFSW, and the abnormal embryo
was developing in pore water plus .044 uM verapamil.
Figure 6. Developmental assay with % abnormal plotted against the
cleavage stage for normally developing embryos as determined by
Newby (11).
Figure 7. Experiment was done and graphed in the same way as the
experiment in figure 3, except that the concentration of verapamil
was doubled.
Figure 8. Developmental assay with % abnormal plotted against the
cleavage stage for embryos with normal development.
Figure 9. Experiment was done and graphed in the same way as the
experiment in figure 5, except that the concentration of emetine was
doubled.
Figure 10. Developmental assay with % abnormal plotted against the
cleavage stage for embryös with normal development.
17
Figure 1.
0.14
0.02
0.00
Rhodamine Assay with Sediment Extract

Ver
+ Ver - SD extr + SD extr
Figure 2.
4/22 Rhodamine Fluorescence Assay with Sediment Extract
0.10

0.08
0.06
004
0.02
0.00
Figure 3.
Examples of Normal and Abnormal Development in S.
purpuratus


A normal embryo at the 16 cell stage.

An abnormal embryo photographed at the same time.
Figure 4.
5/11
puratus Assay with Sediment
100
90-
L
80

A
70-
50
40
30-

20
101
o

k k-
5 10 15 20 25 30 35 40 45 50
time (hours after fertilization)
—2— mfsw
— w
—0— sd. extract 5Oul
——— EtOH (99%) 50 E
Figure 5.
Examples of Normal and Abnormal Development in Urechis
aupo

A normal embryo at the swimming trocophore stage

An abnormal embryo photographed at the same time
Figure 6.
5/19 Urechis Assay with Verapamil (.022 uM)
100
90 -
60

50
0
30

20
107
o
S
6
10
8
cleavage
—— mfsw
—— DW
—0— pw + Ver
er
Figure 7.
5/19 Urechis Assay with Verapamil (.044 uM)
100
90
80-

70-
60
—2— mfsw
—— pw
50
—0— pw + Ver
40
—— + Ver
30-
20
10
S
o

8
cleavage
Figure 8.
5/26 Urechis Assay with Emetine (.0025 uM)
100
90-
80
70
60
50
40
30
20
10
f

—
O +
cleavage
—2— mfsw

Pw
—— pw + Em
— + Em
10
Figure 9.
5/26 Urechis Assay with Emetine (.005 yM)
100
80
70
60
50
10
30
20
10

—
0+
6
8
10
4
cleavage
—— mfsw
— w
— pw +Em
— +Em
Figure 10.
5/26 Urechis Assay with Rhodamine (1.25 uM)
100-
90
80:
70-
50
40
30
20
10
o

6 8 10
cleavage
—— mfsw
PW
— W + R
—— +R