1
THF PRESENCE OF MULTI-XENOBIOTIC RESISTANCE IN MARINE
INVERTEBRATES
ARDIHAU
A multi-xenopiotic resistance p-glycoprotein transport activity
iinked wirn tne multi-drug resistance pnenomenon isolated in human tumor
ceils nas peen tound in various marine invertebrates. Indiviqual cells,
assessed by fluorescence microscopy, possess a Rhodamine B efflux activity
whicn can pe innivitea by Verapamil, a p-glycoprotein transport innibitor.
in addition western piot analvsis indicates the presence a protein of a
similar mojecular weight to the mammalian p-glycoprotein. This activity
appears ro pe a filter feeding lifestyle-linked phenomenon and was found in
Crecnis caupoegqs, Asciaia ceratodeseggs, and Mytilus californianusgil
and mantel tissues. The recognition that marine invertebrates exposed to
man-made and natural noxious substances may possess a multi-xenobiotic
resistance activity is essential to our understanding of organismal
detoxification mecnanisms and the biological impact of pollution and could
be usetil in risk assessment for regulatory policy.
INTRODUCTION
Many marine organisms survive and successfully reproduce in highly
pollured environments Some nave adapted to environments with high
loncentrarions of potentialiv cytotoxic hydrophobic organic compounds
sediment qwellers, for example. reside in conditions wnich concentrate
nuindréds of man-made and natural toxins. (fig. 1) Such marine and estuarian
organisms employ xenopiotic resistance detoxification mechanisms of
which very uttie is understood.
In the past numerous studies on the effects of various pollutants on
marine organisms have been conducted. However, few detoxification
mechanisms nave been identified or are understood. Among these are
cytochrome P-450 detoxification; glutathione con jugation mechanisms;
stress proteins, such as heat shock proteins, and mixed function oxidases
(PFOs). Some of these mechanisms are not active in lower marine
inverteprates In fact, little work can explain which or how many
mecnanisms are employed in many invertebrates.
One recently suggested mechanism is similar to the multidrug
resistance (MÖRi phenomenon seen in some human tumor cells. In mammals
15 known to resuit from selection of celis for cells containing nigh levels
of an FUR protein in response to chemotherapy drug treatment. (fig. 2) This
muifigrug resistance activity is associated with the increaseg proguction of
a memorane giycoprotein, o-givcoprotein (P-gp). This 170 KD
fransmemprane protein nas two ATP binding sites. It prevents the
accuimulation of cnemotnerabeut ic drugs by an energy dependant efflux
mecnanism therepy resisting their toxic effects. (For reviews see Pastan &
otresman. 1991 Endicott ana Ling, 1989; west, 1987; Fairchild and Cowan,
901 kane, Pastan, and Gottesman, 1990.) Similar protein activity has been
solated in rats and other mammals. As yet, its natural "use" or 'role' is
stillunknown
Fuitidrug resistance, has been studied in humans and mammals, but
iffle researen nas examined this phenomenon as a xenobiot ic extrusion
mechanism in tne marine environment. Kurelec and Pivcevic have shown p-
jp linked poilution-resistance activity in musseis (1991) and sponges
Submitrea) in adaition, Barpara Toomey in David Epel's lab has observed
such activity in Urechis caupo 1 am looking for the activity and presence of
p-gp in various marine invertebrates and whether it is correlated with
napirat and/or lifestyle.
My activity assay invoives using fluorescence microscopy to quantify
gifferences in xenobiotic concentration in single cells (oocytes) with and
withouf a p-glycoprotein transport inhibitor. Both fertilized and
unferrilized eggs are incubated in a xenobiotic like fluorescent dye
Rnodamine B with and without Verapamil, a molecule found to bind
specirically to the active site of the P-glycoprotein, thereby inhibiting
pinding of other xenopiotics. After incubation, individual cell fluorescence
was measured In the absence of protein activity the cells fluoresce more.
For organisms tnat snow little or no difference, assumed that xepobjotic
resistance transport activity was minimal or non-existent. (fig. 3)
in addition,! prepared protein samples from various organismal
riesues for protein immunoplots (western blots). Using a mammalian
monocional antipody, C219, selected from hybridomas proguceg from
spienocvtes of mice immunized with SDS-solubilized plasma mempranes of
milltigrug resistant Cninese namster ovary and human ceil lines (kartner,
poreile Bradiev & Ling. 1935) It is among a group of monocional antibodies
which cross react specifically witn tne conserved cytoplasmic domain in
The L-Terminai region of tne ATP binding cassette of the P-gp polypeptide in
a diversity of organisms.
with fürther studies, this information could reveal a novel mechanism
ny which marine organisms survive natur al piant and animal toxins, as well
47 nesticides and otner man-made environment al pollutants. It could
provide insignts into tne evoiutionary origin of this protein, ang may allow
develonment of befrer EPA poilution tests. It might even further our
kliowiedge of numan multigrug resistance activity. It would certaiply help
70 exolain why some organisms are able to survive exposures to a diversity
of xenopiotic compounds.
ATERIALS & FIETHODS
ORGANISFIS - collection and maintainance:
All organismns were kept in tanks equiped with a flowing sea water pumping
sustem
ascloia ceratoges The tunicates were collected from the underside of
Tioare in Honterey marina
Mamys nastata, Henricia jeviuscula, Patiria miniata These organisms
were nonared ny Plonterey Bay Aquarium
ongaster excentricus, rucrociona Proiifera. Mytiius californianus, Ternva
mrlim Tne sand goilars sponges, and mussels were previously
ollected from Ponterey Bay and kept in tanks. They were donated by the
staft of Honkins Harine Station
deiiraosus pregonensis. Macoma nasuta Crechis caupa These organisms
were collected from tne muaf lats of Ploss Landing Harine Harbor.
Lyrechiniis bictus. Strangylocentrotus purpuratus: The sea urchins were
previousiy collected by the Epel lap ang stored in tanks
rslibe leonina inese nutabrancs were collected from the kelp pegs of
onerey ay
FLUURESLENCE ASSAV
spawning of eggs and sperm
onqviocentrotus purpuratus, Lytechinus pictus, Dendraster excentricus
Tne sea urchins were spawned using standard procedures. (Johnson and Epel.
985) Tne organisms were injected with approximately 0.5-2cc 0.5 MKCI.
depending on the individual's size. The needle was inserted diagonally
outward along the sides of the coelômic cavity to induce spawning from the
gonads Snaking the injectea organism often facilitated spawning.
Subsequently, the organism was positioned with the aboral surface
immersed in filtered sea water (FSW) when collecting eggs. If the gametes
were sperm. they were suctioned off using a glass pipet. The eggs were
keht in a constantly stirring dilute solution with FSW at 162c. The
concentrated sperm were refrigerated in an Eppendorf tube.
ernis cape lo prepare the spawning insturment, the center of a
cabillary rübe was thinned using a bunsen burner to melt and stretch the
mindie ine tube was proken into two pieces and the proken eng was
remeited to form a small qull bulb. The thin end of the prepareg capillary
rüne was inserted into one of tne four jower gonapores on the brechisand
moved pack and forth gently in a rhythmic fashion a few times. Stroking
Ubward toward the gonavore often facilitated spawning. The reddish eggs
were wasned off of the female witn a glass pipet and FSW into a beaker of
row kenf at 1620 The milky sperm concentrate was suctioned off with a
qlass pipet and refrigerated in an Eppendorf tube.
asrioia ceratodes These invertebrates are seif-sterile hermapprodites.
Scissors inserted at the distal of two pores were used to cut the tunicates
enigermis It was peeled from this outer skin and the eggs and sperm could
e seen in distinct sacs. Using a moist glass pipet the eggs were suctioned
ouf of the sac and into FSw kept at 162C. The sperm were similarly sucked
ub and stored in an Eppendorf tube.
Preparation of eggs for fertilization:
ne Stronqviocentrotus purpuratusegq suspension was passed through a 90
mm nyrex mesn five or six times to remove the jelly layer. The eggs were
Then nand centrifuged and the supernatant was suctioned off, after which.
The eggs were resuspended in FSW. The Lytechinus pictusegg suspension
was similariy passed througn a 120 mm nytex mesh for de jellying. The
Gengraster excentricuseggs were hand centrifuged multiple times to
remove follical cells. As this did not work, the eggs were suspended in a pH
Fow solution prepared with 1 MHCI followed by multiple centrifugation.
if is not clear whether this is harmful to the eggs.) The Ascidia ceratodes
euns were not separated from their surrounding follical cells.
reraro
-reirigerated sperm concentrate is potent for 3-5 gays. A sperm dijution
was brepared trom 1-2 ml of FSw and 1 drop of concentrateg sperm. One
roo of sperm dilution was agded to a 1O ml eag suspension ang swirled.
Arter abproximatelv 5 minutes a grop of the fertilized eggs were observed
under the microscope for a good fertilization membrane. Howeyer, in the
ascinian egge it was not possiple to detect if fertilization hag occurred
until affer first cieavage, so the eggs were used assuming fertilization was
Formai The sperm were wasned out by nand centrifugation ang suctioning
aff of the siipernatant. The eggs were then resuspended into Fsw.
A5V
Oul of a ImH solution of Rhodamine B was added to 1Oml of egg suspension
for a luri rinal concentration. The eggs were divided into 2-5m1 aliquots.
and 25ul of zuh verapamil were added to one aliquot for a lOug/ml final
concentration Tne samples were incupated for 1 hour at 162C, hand
centrifuged, washed out the Rhodamine supernatant, and then resuspend in
-2miFSw. Tne cells were immediately positioned under a 40X lens of an
epifluorescence microscope and the aperature was closed to a fixed point.
Fluorescence measurements were taken on 10 individual healthy eggs by a
phofo sensing device attacned to the microscope. Using those readings
means and standard deviations were calculated. The above procedure was
followed for both fertilized and unfertilized eggs for all organisms for
which oocvres were availaple.
WESTERN BLOTS
prorein sampies.
rinely cnopped rissues trom various organisms were placed in Eppendorf
tübes from wnich proteins were extracted witn 5% sogium dodecyisulfate
) wirn rne protease innipitors 1OuM phenyl-methyisulfonyl flourige
(Prs Imin soybean trypsin inhibitor, and I mM EDTA. The samples were
sonicared and centrifuged after 1 hr. of solublization, supernatants
fransferred to new Eppenaorf tubes, and the remaining pellet discarded.
Prorein concentrations were obtained by preparing 1.20, 1.50, or 1:100
iutions of each sample. 5 ul samples of each dilution were placed in
triplicare on a 96 weil micro-titer plate. A bicinchonic acid (BCA) assay
ution was prepared and löoul of the BCA/copper sulfate solution was
reacted witn each dijution sample overnignt. The protein concentration was
read using a Molecular Devices VHax plate reader at 560 nm.
Gel eiectropnoresis and western piotting.
Samples were loaded onto a 7 5% polyacrylamide gel at approximately zoug
per lane The proreins were separated on a BioRad minigel apparatus using a
fris/giveine running puf fer and transferred to nitrocellulose membranes
Using à BioRag electrophoretic transfer system. The transfer occured over
voitage agjusted to 13 v for 1 hour, 24 V for 3 hours, and 36 V overnignt.
ne biots were blocked with PBS-Tween 20 (0.052)- BSA (10mM) for a half
hour and then incubateg for 3 hours with C219 monoclonal antibody (1:1000
in PBS- ween 20-BSA) commercially available from Centocor. The
nitrocellulose membrane was washed for 10 minutes with 1Omls of the
PRS-Iween 20-PBS blocking solution three times and then incubated for1
nour with goat anti-rapbit alkaline phosphatase con jugated antibody (Sigma)
1000 in PBS-Tween 20-BSA). It is then washed three times for 10
minutes each witn 10 mi of PBS-Tween 20 (0.057) and incubated in apH 9.5
nurfer 1100 mM Tris, löömH Nacl, SmM Maclz) with nitro-biue tetrazolium¬
-Cromo--chioro-3 indolvi onosphate for color gevelopment. (Bragley &
war )
HSULTS
FLOURIMIETRIC ASSAY
Urechis caupoand Ascidia ceratodeseggs show a transport act ivity
characteristic of MDR activity. As seen by figures 4 and 5, eggs incubated
only with Rhodamine B exhibit less fluorescence then those incubated in dve
plus verapamil, the MIDR activity inhibitor. Vrechiseggs normally contain
0% -25% of the Rhodamine B concentrated by eggs in which the transport
activity is inhibited. Ascidianeggs normally concentrate only 252 - 502 of
that in with which transport activity is inhibited.
Experiments conducted on Strongylocentrotus purpuratus, Lytechinus
oictus and Dendraster excentricusshowed no detectable MDR transport
activity. As shown by figures 6 and 7, there is little difference between
samples incubated with and without Verapamil. Therefore these organisms
do not appear to nave the MDR activity.
PTIUNOBLOTS
Western blots on Urechis caupoeggs, Ascidianeggs, and Mytilus
calirornianisgill and mantle tissue showed a positive cross reaction with
the C219 mammalian monoclonal antibody. The bands were 142 KD. 146 kD.
and 138 ku respectively, indicating that these organisms have a protein that
is aifferent, though close in molecular weight to the 170 kD mammalian p¬
qivcoprotein responsible for MDR activity. In addition the Mtilus gill and
mantel tissue and the Asciaianeggs had cross reacting bands for proteins of
different molecular weights. The mussel had a 200 kD band and both had 34
KU pangs.
Strongylocentrotus purpuratustissue and eggs, Lytechinus pictus
tissue and eggs, Denaraster excentricustissue and eggs, Patiria miniatagut
tissue, Henricia leviusculagut tissue, Ascidia ceratodestissues, Urechis
tissues, lethva aurantium and Hicrociona proliferadid not show ant ibody
recognition, indicating a lack of a similar cross-reacting protein.
Hemigrapsus oregonensis Macoma nasuta, Chlamys hastata, and
Metibe leoninasnowed cross reactions at different molecular weights that
were smudged. It is not clear whether there is a distinct protein.
DISCUSSION
The results indicate MDR like transport activity in Urechiseggs,
Ascidianeggs, and muscle tussue. Such activity does not appear to be
present in echinoderm eggs. Although the evidence is limited, it suggests
that MOR like transport activity is correlated with a filter feeding lifestyle
Fluorescence assays and Western blots indicate that the sea urchins,
S purpuratus and L. pictus and the sand dollar D excentricusdo not have
PIDR transport activity and do not have cross-reactivity to the C219
antibody. The sea stars P miniataand H leviuscula do not possess a
related protein and it is unclear whether the clam M nasuta the crab H
oregonensis or the scallop C hastata have a cross-reacting protein.
Altnough the C219 monoclonal antibody does not show cross-reactivity in
tne marine sponges Tethva aurant ium and Microciona prolifera it is again
unciear as to whether there is similar protein activity, due to conflicting
evigence by Kurelec and Pivcevic (in press). Finally, fluorescence activity
assavs and western blots clearly indicate the presence of an MDR like
transport activity in the mussel Mytilus californianus the tunicate Ascidia
ceratodes and Urechis caupa
As both, the fluorescence assay indicated activity and the Western
blot resulted in a positive cross reaction, it is possible to conclude that
Urechis caupoand Ascidia ceratodeshave a MDR-like gene which produces a
multi-xenobiotic resistance protein in its eggs. However, as Iwas unable to
defect tne presence of the protein in adult tissue, the organism may stop
proaucing the protein if it is no longer necessary. Both of these organisms
nave a thick epigermis that may serve to protect it from toxins, thereby
making the protein obsolete in the adult. Also, due to problems with
smudging in the western blots, organismal gut tissue needs to be retested
to form conclusions. Both of these organisms are filter feeding and reside
in toxin filled environments.
Mytilus californianisgill and mantle tissues reacted positively with
the C219 monoclonal antibody for a 138 kD protein. As the mussel was past
its spawning season ! was unable to conduct assays on its oocytes. This
filter feeder also showed cross reactions at higher (200 KD) and lower (34
kD) molecular weights. This could be because more than one protein with a
similar sequence and/or function is present, though lower molecular weight
bands are most likely the result of protease degradation.
Negative fluorescence assay results from sea urchin and sand dollar
eggs indicate that as these organisms show no differences in fluorescence,
there appears to be no transport protein activity of Rhodamine B. Also,
negative results on the immunoblots for these organisms and sea stars
indicate that the protein is not present in the organisms. Though the results
are not conclusive in themselves, they lead me to believe that echinoderms
do not possess a MDR like transport protein.
Assay attempts on the marine sponges Microcionaand Tethyawere
inconclusive. Individual cell dissociation, though possible, are very
difficult as many other symbiotic organisms live in sponge tissue and must
be separated from the sponge cells. In addition, individual cells were too
small to recora conclusive fluorescence readings due to high background
fuorescence interference. Despite these problems, the results from these
say attempts gave positive activity indications. Western blotting, on the
ofher hand, showed no cross reaction, though, Kurelec and Pivcevic from
vugoslavia have detected an MDR like 125 KD protein in Tethya with a
polyclonal ant ibody (submitted) and have noted xenobiotic resistance
activity in tissue with radiolabelled drugs (submitted). This conflicting
evidence may be explained by the fact that the sponge is an extremely
primitive organism as compared to the mammals from which the C219
monoclonal antibody is created and that may be too specific, as this MDR¬
like transporter is likely to have evolved significantly. This also indicates
that other organisms have screened may also possess such a protein that
is not detectable with the C219 monoclonal antibody.
Immunoblot cross reactions at different molecular weights indicate
that there is likely to be a protein similar to the MDR p-glycoprotein. As
the monoclonal antibody is specific for the ATP binding cassette in the C
terminal region of the 170 kD mammalian MDR P-gp, it is quiet specific.
However, it is still general enough to have some degree of error by cross
reacting to other protein ATP binding cassettes with similar sequences.
Therefore, à negative cross reaction, though somewhat inconclusive has high
propability of indicating lack of protein. Ambiquous (smudged) positive
cross reactions may be the result of a cross reacting protein that had been
dégraded, put without a clear band are inconclusive. Positive cross
reactions at molecular weights differing from the 170 KD mammalian p¬
giycoprotein can be explained by evolutionary differences across phyla in a
protein with similar function as the mammalian MDR p-glycoprotein. Lower
molecular weight bands such as in Hemigrapsus oregonensisor Ascidian
eggs, could be the result of protease degradation of a larger protein.
Attempts at using protease inhibitors such as EDTA, PMSF, and soybean
trypsin innibitor appeared not to be successful in preventing protein break
down if it had occurred. However, as isolation of the active protease is
often difficult, 1 may not be inhibiting the correct protease.
It was not possible to conduct fluorescence assays on individual cells
of many organisms as it is difficult to get gametes or dissociate tissue. In
some cases, the organism was not gravid or was past its spåwning season.
In others, the cells were not usable with this assay. For example, mediaster
produces eggs that are naturally fluorescent thereby making individual
fluorescence readings meaningless. Also, sponge tissue cells were too
small to conduct quant if iable fluorescence readings without high amounts
of background fluorescence interference
My results indicate that multi-xenobiotic resistance activity is
associated with filter feeders. The Urechis caupo, Mytilus californianis,
and Asciala ceratodesare all filter feeders that habitate polluted
environments. Vrechig a sediment dweller, is exposed to concentrated
man-made and natural noxious substances. Mytilus californianusis also a
resident of the sediments. The Ascigianis found under floats in Monterey
Marina wnich is depositea with pollutants. The echinoderms, on the other
hand live in sandy/rocky, relat ively unpolluted habitats, and may not need
and tnus nave not developed such xenobiotic resistance activity.
ACKNOWLEDGEMENTS
would like to thank my advisor, Dr. David Epel for his encouragement
and worgs of wisdom when frustration may have been detrimental. would
also like to thank Barbara Holland-Toomey, who has been my mentor and
taugnt me a great deal. Dennis LaRochelle and Rob Swezey were invaluable
for generously sparing their vast knowledge. In addition, Chris Patton can
not be thankea enough for keeping me sane and helping with his infinite
wizardry, creativeness, photography, words of advice, pretzels/chocolate,
and a ready smile. I would like to thank my lab mates Leonard D'Amico, Jeff
Jackson, and Steve Verbinsky for being always available distractions when
needed them most. Also, Chris Harrold and See Listen from Monterey Bay
Aquarium for their generous donations of organisms. Thanks to all of the
staff of Hopkins Marine Station for donating their various invertebrates.
And last but not least, many thanks to the members of the Thompson lab who
often fed (especially their plentiful, pick me up in the morning and
afternoon, herbal tea), encouraged, listened, inspired, and often distracted
me. And thank you to Mandy Schivell, my roommate and forever friend, and
the members of the Hopkins luxury triplex.
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Gerlach, J.A., et. al. (1986). Homology between P-glycoprotein and a
bacterial hemolysin transport protein suggests a model for multidrug
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Gros, P., J. Croop, and D. Housman (1986). Mammalian multidrug resistance
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