Erica Li
Bio 175H: Problems in Ecology and Ecophysiology
Advisor: Epel
Bisphenol A causes disruptions in the cell cycle of the zygotes and
embryos of the purple sea urchin, Strongylocentrotus purpuratus
Abstract
Bisphenol A (BPA), a common chemical found in polycarbonate plastic
containers, is suspected to damage animal health. While many studies on BPA have been
done on terrestrial vertebrate models, few have been conducted on marine invertebrates
Because BPA is found in harbours, it is important to find out if we need to be concerned
about BPA pollution of the marine ecosystem. This study examines the effects of BPA
on the early development of the purple sea urchin, Strongylocentrotus purpuratus. The
results show that the vast majority of the embryos develop normally when exposed to
concentrations at or below 1 uM up to the late prism stage; a few exogastrulating
embryos appear as a result of BPA exposure, but the number of exogastrulating embryos
does not increase with increasing concentration. BPA inhibits formation of the blastocoel
in S. purpuratus embryos at concentrations greater than 1 uM. At concentrations greater
than 5 uM, BPA significantly delays cell division, apparently affecting the mitotic
apparatus of the zygote and preventing chromosomes from condensing and migrating
properly. The concentrations used in this study are orders of magnitude higher than what
has been found in the environment, indicating that BPA may not be of concern in marine
ecosystems.
Erica Li
Bio 175H: Problems in Ecology and Ecophysiology
Advisor: Epel
Bisphenol A causes disruptions in the cell cycle of the zygotes and embryos
of the purple sea urchin, Strongylocentrotus purpuratus
Abstract
Bisphenol A (BPA), a common chemical found in polycarbonate plastic containers, is
suspected to damage animal health. While many studies on BPA have been done on terrestrial
vertebrate models, few have been conducted on marine invertebrates. Because BPA is found in
harbours, it is important to find out if we need to be concerned about BPA pollution of the
marine ecosystem. This study examines the effects of BPA on the early development of the
purple sea urchin, Strongylocentrotus purpuratus. The results show that the vast majority of the
embryos develop normally when exposed to concentrations at or below 1 uM up to the late prism
stage; a few exogastrulating embryos appear as a result of BPA exposure, but the number of
exogastrulating embryos does not increase with increasing concentration. BPA inhibits
formation of the blastocoel in S. purpuratus embryos at concentrations greater than 1 uM. At
concentrations greater than 5 uM, BPA significantly delays cell division, apparently affecting the
mitotic apparatus of the zygote and preventing chromosomes from condensing and migrating
properly. The concentrations used in this study are orders of magnitude higher than what has
been found in the environment, indicating that BPA may not be of concern in marine
ecosystems.
Introduction
Bisphenol A (BPA) is gaining attention for its ability to interfere with the health of
animals. It is a common industrial chemical, found in polycarbonate plastics that are used for lab
flasks, animal cages, and many consumer products such as drinking water jugs and bottles.
(Colborn et al, 1996) As a xenoestrogen, BPA is many orders of magnitude less potent than
estradiol. (Milligan et al, 1998) However, according to David Feldman, a professor of medicine
in the endocrinology division of the Stanford School of Medicine, it "still has activity in the parts
per billion range." (Colborn et al, 1996) BPA is also present in relatively high concentrations-
as high as eighty parts per billion—in canned foods, such as canned corn, artichokes, and peas.
(Colborn et al, 1996)
A number of studies have been done on terrestrial vertebrate model organisms to
ascertain the health-damaging effects of BPA, some with seemingly conflicting results. Masaru
Furuya et al showed that dietary BPA at 200 mg per rooster per week feminizes roosters. (Furuya
et al, 2003) According to a multi-generation study conducted by Rochelle Tyl et al, dietary BPA
did not significantly damage the health of Sprague-Dawley rats or the health of their offsprings.
(Tyl et al, 2002) A study on mice by Patricia Hunt et al, however, demonstrated that BPA causes
meiotic aneuploidy in the oocytes of female mouse. (Hunt et al, 2003) Possibly related to this,
Lehmann and Metzler concluded that Bisphenol A interferes with microtubules in cultured
human fibroblasts. (Lehmann and Metzler, 2003) In a study on a variety of chemicals suspected
to cause aneuploidy, Parry et al demonstrated that bisphenol A induces aneuploidy in Chinese
hamster V79 cells at 14 ug/ml. There was a dose-dependent increase in the frequencies of both
abnormal metaphases and all mitotic aberrations when those cells were exposed to BPA. Parry
et al concluded that BPA may interact with components of the centrosomes which make up the
poles of the mitotic spindle. (Parry et al, 2002)
Less attention has been paid to how BPA affects organisms in aquatic systems. In one
study by H. Segner et al on the freshwater zebrafish, Danio rerio, BPA was shown not to be a
potent xenoestrogen. (Segner et al, 2003) However, no BPA study has been conducted on
marine invertebrate model organisms, despite the fact that a nationwide investigation of
endocrine disruptors in Japan’s harbors, conducted by Hosokawa et al, found BPA in at least
thirty percent of the sites surveyed. (Hosokawa et al, 2003)
It is thus relevant to examine the effects of bisphenol A on a marine invertebrate model
organism in order to ascertain whether the scientific community and policy makers need to be
concerned about BPA pollution of marine systems. The studies by Hunt, Lehmann and Metzler,
and Parry suggest that it is worthwhile to examine the effects of BPA on cell division. The
experiments described below were designed to examine how BPA affects cell division and
differentiation in the early development of the sea urchin, Strongylocentrotus purpuratus. The
results show that BPA at concentrations above 5 uM disrupts S. purpuratus development by
affecting mitosis even at the fertilized one-cell stage. Embryos exposed at 3 uM of BPA divide
slower than the controls and never form the blastocoel, possibly due to disruptions of the mitotic
apparatus. In contrast, embryos exposed to 1 uM of BPA or less develop normally till at least
the late prism stage.
Materials and Methods
General protocols common to all three parts of the experiment
A O.1M stock solution of bisphenol A was made in DMSO, stored at room temperature in
a covered graduated cylinder, and shielded from light with tin foil. All dilutions of the stock
solution were done using filtered seawater.
Sea urchins (Strongylocentrotus purpuratus) were induced to spawn by injection with
approximately 1 mL of O.5M potassium chloride into the coelomic cavity. Females were placed
upside-down on top of a small beaker (beaker diameter smaller than the diameter of the urchin)
filled with sea water, such that eggs fell to the bottom of the beaker. The eggs were stored in the
cold room at 16 °C and could be used for about two days. Sperm was pipetted from the dorsal
side of the males directly into an eppendorf tube and stored in the refrigerator at 4 °C.
The density of zygotes in the culture was low such that the zygotes would not get anoxic
To achieve this, about 0.2 mL of super-concentrated egg suspension was diluted in a beaker
containing 500 mL of filtered sea water. Fifteen minutes after 5 uL of sperm was added to the
eggs in BPA-free sea water, the eggs were checked under the microscope to see if they possessed
the fertilization envelope. If more than 90% of the eggs were fertilized, they were exposed to
control and experimental conditions, described below.
Part one: does BPA affect the developmental schedule of S. purpuratus embryos up to the
late prism stage?
Fertilized zygotes were placed in covered glass petri dishes containing 10 mL of sea
water and various concentrations of BPA ranging from O.1 uM to 1 mM. The control group was
kept in the same kind of petric dishes with the same volume of sea water, but without any BPA.
Sperm from three males were used to fertilize the eggs of one female. Thus, for each
concentration of BPA, there were three samples total. The zygotes were checked by light
microscopy at 15 minutes, 2 hours, 4 hours, 6 hours, 20 hours, 23 hours, 46 hours, 49 hours, and
70 hours after fertilization. In between sampling, the embryo cultures were left in a cold room
maintained at 16 °C.
When this part of the experiment was repeated, fertilized zygotes were placed in glass
test tubes that were covered with parafilm. The test tubes rested on a rack in the 16 C cold
room.
Part two: does BPA affect the cell division schedule
The experiment was conducted in glass test tubes containing control, 1 uM, 3 uM, 5 uM,
and 7 uM of BPA. The other conditions were the same as those described in Part One. Pure
DMSO was diluted 100 times with filtered seawater and added to the control group, such that the
control group had the same amount of diluted DMSO as the sample with the highest amount of
BPA. Fertilized embryos were added to each test tube and checked using light microscopy about
every 30 minutes until the control group arrived at the morula stage.
Part three: BPA’s effect on chromosome appearance during the first cleavage
A solution containing 7 uM of BPA was made. Eight Falcon polystyrene test tubes were
filled with 10 mL of control samples each. Another eight tubes were filled with embryos
exposed to 7uM of BPA. The 16 tubes were placed on a rocker table for 1 hour at room
temperature. Then, every 15 minutes, one control and one experimental sample were taken and
centrifuged in a hand-crank low-speed centrifuge such that the embryos settled at the bottom of
the test tubes. The liquids from the two tubes were aspirated. 5 mLs of Carnoys solution (1 part
glacial acetic acid: 3parts ethanol) was added to both tubes, and the samples were placed on a
test tube rack at room temperature. This process was repeated until all eight sets of zygotes were
fixed in Carnoys.
After approximately 24 hours, the Carnoys solution was aspirated from all test tubes. All
zygotes were placed in 45% acetic acid for about 30 minutes. The acetic acid was then removed
and a solution of 2% acetocarmine with 45% acetic acid was added to all samples. After 10
minutes, the cell samples were placed on a slide under a phase microscope and examined for
chromosome arrangement.
Results
Part one: does BPA affect the life stage schedule of S. purpuratus embryos up to the late
prism stage!
A vast majority of S. purpuratus embryos were able to grow to at least the late prism
stage in BPA concentrations less than or equal to 1 uM. At concentrations greater than 3 uM,
however, 100 % of the embryos failed to develop a blastocoel and to make it past the morula
stage. At concentrations above 25 uM, embryos lysed and died early at the 1-cell stage.
Concentrations of 3 uM, 6 uM, and 10 uM caused development to be delayed. Problems
with cell division were seen in the 3 uM group at 2 hours after fertilization, when the zygotes
were going through the first cleavage. Many of the zygotes exposed to 3 uM had various
problems such as asynchronous cell division, wrong number of cells, and incomplete cytokinesis.
The developmental progress of the 6 uM, and 10 uM groups was delayed further than the 3 uM
group, and the morphological abnormalities were more prominent. At 2 hours after fertilization,
there were some normal-looking zygotes at the 2-cell stage in the 3 uM group, but none in the
groups exposed to 6 uM of BPA or more.
Even though embryos exposed to 3 uM, 6 uM, and 10 uM of BPA displayed delays and
abnormalities at 2 hours after fertilization, their cells continued to divide abnormally and many
survived till at least the third day after fertilization. At 76 hours, some embryos exposed to 10
UM of BPA were swimming, even though they were dumbbell shaped and completely abnormal
There were no exogastrulates in the control groups. In all experimental groups up to 1
UM, there were a few exogastrulating individuals. The number of exogastrulates and the degree
of exogastrulation did not seem to vary significantly with varying concentrations.
Part two: does BPA affect the cell division schedule
Given the above observation that BPA concentrations at greater than 3 uM delay
development, a study was conducted to see if BPA delays cell-division. It was observed that at
about 2 hours after fertilization, the control, 1 uM, and 3 uM groups were all predominantly at
the 2-cell stage. The cell division schedule in the 5 uM group was delayed—one could see the
cells in the process of dividing but cytokinesis had not yet happened at 2 hours. In the 7 uM
group at this time, all zygotes were completely at the 1-cell stage, looking round.
At about 2.5 hours after fertilization, the control and 1 uM groups looked mostly healthy.
Both groups had approximately equal numbers of 2-cell and 4-cell stage embryos. Cell division
abnormalities started emerging in the 3 uM group at this time, though many of the embryos also
made it to normal 2-cell and 4-cell stages. The 5 uM group was clearly developmentally behind
the groups with lower concentrations. Most of the embryos exposed to 5 uM BPA were still at
the 1-cell or 2-cell stages, and the ones that were beyond the 2-cell stage clearly had incomplete
cytokinesis and unequal divisions. (Fig. 1) In the 7 uM group, there were hardly any discernible
boundaries between cells. Most of the zygotes could be categorized as being at the 1-cell stage,
but there were invaginations in the cell membrane and the zygotes were no longer round. Each
embryo in this group contained fewer cells than the embryos of the other groups.
At about 3.5 hours after fertilization, the control group was predominantly at the 8-cell
stage. The 3 uM and 5 uM groups had predominantly abnormal zygotes with 8 or more cells.
The 7 uM group, in contrast, had mostly morphologically abnormal zygotes that clearly had less
than 8 cells. At about 4 hours after fertilization, the control, 1 uM, and 3 uM groups were
apparently at the morula stage, whereas the 5 uM group contained comparatively less cells and
more deformities. The 7 uM group was delayed even further. It had more cases of incomplete
cytokinesis and more morphologically deformed cells than the 5 uM group.
Later on, all the zygotes looked like they were at the morula stage and it became
impossible to visually discern how many cells an embryo had. The sheer number of cells also
made it impossible to clearly discern the morphology of the cells, and all the samples started
appearing similar to one another. At this point, the experiment was halted.
Part three: how does BPA affect cell division
In this third study, I examined in more detail the inhibition of cell division. At first the
control and experimental group appeared similar. (Fig. 2 and 3) At about 1.75 and 2 hours after
fertilization, the chromosomes in some of the zygotes in the control group began to organize in
two sets, lined up, and migrated to one side of the zygote. The chromosomes of all zygotes in
the experimental group still appeared scattered and more near the center of the zygote. (Fig. 4)
At 2.25 and 2.5 hours after fertilization, some of the zygotes in the control group showed clear
division and arrived at the 2-cell stage, whereas no 2-cell stage zygotes could be found in the
experimental group. In the control group, two sets of chromosomes could be seen in some
zygotes, but in all experimental zygotes only one set of chromosomes could be seen. (Fig. 5 and
The result from this part of the experiment was problematic in that not all zygotes in the
control group divided. In the samples where the zygotes did divide, the cleavage furrow was
often difficult to discern, making visualizing two sets of chromosomes the only indication of cell
division. Oftentimes, only one set of chromosome was found in the zygotes of the control group,
which was possibly confounded by the fact that the fixing and staining technique described did
not yield extremely clear images of the chromosomes. Nonetheless, there was a difference in
chromosome morphology between the control and experimental groups. It was significant that
no zygote in the experimental group divided in 2.5 hours, whereas at least some divided in the
control group.
Discussion
The experiments demonstrate that S. purpuratus embryos are able to develop normally up
to the late prism stage in BPA concentrations up to 1 uM. At concentrations equal and above 3
UM, BPA delays development, inhibits blastocoel formation, delays cell division, and affects
chromosome arrangement during mitosis. However, concentrations up to 10 uM of BPA are not
immediately lethal, and abnormal embryos continue to experience cell division despite the
failure to form the blastocoel until at least 70 hours after fertilization.
The individual exogastrulates are difficult to explain. None existed in the control group.
but the number of exogastrulates did not increase significantly with increasing concentration. As
the exogastrulates were greatly in the minority, it is difficult to draw conclusions regarding the
effects of BPA on exogastrulation. It would be helpful to repeat each part of the experiment a
few more times and perform them in a more streamlined, consistent way.
Compared to past studies and publications, some of the results from this study are
surprising. Hunt et al concluded that “low-dose BPA exposure... is sufficient to cause meiotic
abnormalities." (Hunt et al, 2003) Hunt et al fed the mice bisphenol A at 20, 40, or 100 ngg
body weight/day for 6-8 days preceding oocyte analysis. To determine the shortest exposure that
produced detectable effects, another set of experiments using a dose of 20 ng BPA/g body
weight for 3, 5, or 7 days prior to oocyte analysis was conducted. From these experiments, Hunt
et al concluded that BPA causes aneuploidy in the oocytes of female mice even at low doses.
In my experiment, I cannot feed BPA to sea urchin embryos using ng BPA/ body weight
because the embryos are too small. For the purpose of our discussion, we shall use the weight of
the sea water surrounding the embryos in the denominator and assume that an embryo's body is
also predominantly water. The molecular weight of BPA is 228.29. Calculations show that if
Hunt et al fed BPA to mice at 20 ng BPA/ g mice body weight, then they were feeding 8.76 x 10
moles of BPA to each kilogram of mice. Assuming the density of animal body and water are
approximately the same, the concentration at which Hunt et al achieved an effect was 8.76 x 10
M. The lowest concentration where my experiment achieved a clearly discernible effect was 3
x 10° M. Thus, Hunt’s effective concentration was about 33 times lower than mine. However,
because BPA is practically insoluble in water, it may accumulate in the fatty tissues of the mice.
This may explain the extremely low effective concentration presented in Hunt et al’s study.
Remarkably, in the study conducted by Parry et al, an effectively aneugenic concentration
of BPA was as high as about 40 uM. (Parry et al, 2002) At such concentration, Parry et al
showed that dividing cells became multipolar because the chemical may have interacted with
components of the centrosomes that make up the poles of the mitotic spindle. In contrast to
Parry et al’s finding, my experiments show that 40 uM of BPA would kill the S. purpuratus
embryos instantly. In addition, according to Tyl et al, BPA damages the health of SD rats at only
above 750 ppm. (Tyl et al, 2002) This concentration is equal to 3.285 x 10 M, which, like
Parry's effective concentration, is significantly higher than the concentration that inhibits
blastocoel formation in sea urchin embryos.
The wide differences among effective concentration shown by the aforementioned studies
suggest that there may be species-dependent factors regarding vulnerability to BPA. The levels
of BPA that affect mice, rats, and hamsters, three species of rodents, are completely different.
11
Furthermore, concentration that affects the early development of sea urchins is different from the
concentrations that affect any of the rodent species in the aforementioned studies. Because
marine life is extremely diverse, one must take care when attempting to extrapolate the sea
urchin embryo data to other species.
The environmental relevance of the concentrations used in my sea urchin embryo study
should also be discussed. In an environmental study on BPA, the brother and sister team of
Fatima Olea, a food toxicologist, and Nicolas Olea, a physician specializing in endocrine
cancers, found BPA in canned vegetables at 80 parts per billion. "At such levels," wrote Theo
Colborn and her co-authors of Our Stolen Future, “a synthetic estrogen mimic might contribute
significantly to a person’s exposure regardless of whether it is a "weak" estrogen or not.'
(Colborn et al, 1996) 80 ppb of BPA was cause for human health concerns. However, assuming
that canned vegetables are mostly made of water, 80 ppb of BPA translates into a concentration
of 3.504 x 10 M, nearly an order of magnitude lower than the concentration that inhibited
blastocoel formation in S. purpuratus embryos.
While the report by Hosokawa et al did not mention the concentration of BPA that was
found in harbors, it is unlikely that marine concentrations of BPA would exceed that found in
manufactured foods. Thus, the fact that 3 uM of BPA inhibits blastocoel formation in sea urchin
embryos may not be environmentally relevant, as marine life forms are probably not likely to
encounter such high concentrations. Sea urchin embryos show considerable tolerance to 1 uM of
BPA up to the prism stage, demonstrating the robustness of early embryos. However, it is
unclear whether early exposure to BPA would affect later development. It would be important to
conduct future experiments by using lower concentrations, by culturing the embryos to later life
stages, and by using other marine model organisms.
This study is one of the few that focuses on the effects of bisphenol A on aquatic
organisms, and possibly the first that explores the effects of BPA on the early development of a
marine invertebrate species. The findings raise questions regarding the action of BPA on
different model systems, and prompt further investigation on the mechanisms and concentration
ranges through which BPA affects animal and human health.
Acknowledgements
Thank you, Professor Epel, for your guidance, patience, resource books, inspiration, and
butt-kicking.
Thank you, Rebecca Vega, for all your patience when I interrupt your work, and for
your suggestions and demonstrations.
Thank you, Amro Hamdoun, for teaching me the lab etiquettes.
Literature Cited
Colborn, Theo, Dianne Dumanoski, and John Peterson Myers,1996. Bisphenol A. pp.
130-131, 135 in Our Stolen Future. Penguine Books USA.
Czihak, G. 1975. The Sea Urchin Embryo: Biochemistry and Morphogenesis. Springer-
Verlag Berlin Heidelberg, New York.
Furuya, Masaru; Fumihiko Sasaki, Amin M.A. Hassanin, Sachi Kuwahara, Yasuhiro
Tsukamoto. 2003. Effects of bisphenol-A on the growth of comb and testes of male
chicken. Can J Vet Res. January 2003; 67 (1): 68-71
Hunt, Patricia, Kara E. Koehler, Martha Susiarjo, Craig A. Hodges, Arlene Ilagan, Robert
C. Voigt, Sally Thomas, Brian F. Thomas, and Terry J. Hassold. 2003. Bisphenol A
Exposure Causes Meiotic Aneuploidy in the Female Mouse. Current Biology, Vol. 13,
546-553.
Lehmann, L.; Metzler, M. 2003. Bisphenol A interferes with microtubules in cultured
human fibroblasts. Toxicological Sciences; v.72, no.S-1, p.254.
Milligan, S. R., Balasubramanina, A. V., and Kalita, J.C. 1998. Relative potency of
xenobiotic estrogens in an acute in vivo mammalian assay. Environmental Health
Perspect. 106, 23-26
Parry, E.M.; J.M. Parry, C. Corso, A. Doherty, F. Haddad, T.F. Hermine, G. Johnson, M.
Kayani, E. Quick, T. Warr and J. Williamson. November 2002. Detection and
characterization of mechanisms of action of aneugenic chemicals. Mutagenesis, Vol. 17,
No. 6, 509-521.
Segner, H.; Navas, J.M.; Schaefers, C.; Wenzel, A. March 2003. Potencies of estrogenic
compounds in in vitro screening assays and in life cycle tests with zebrafish in vivo.
Ecotoxicology and Environmental Safety; v. 54, no. 3, pp. 315-322
Tyl, R.W., C. B. Myers, M.C. Marr, B.F. Thomas, A.R. Keimowitz, D.R. Brine, M.M.
Veselica, P.A. Fail, T.Y. Chang, J.C. Seely, R.L» Joiner, J.H. Butala, S.S. Dimond, S.Z.
Cagen, R.N. Shiotsuka, G.D. Stropp, and J.M Waechter. 2002. Three-Generation
Reproductive Toxicity Study of Dietary Bisphenol A in CD Sprague-Dawley Rats.
Toxicological Sciences 68, pp. 121-146.
Yokota, Yukio.; Valeria Matranga; Zuzana Smolenicka. 2000. p. 84, pp. 206-207 in The
Sea Urchin: From Basic Biology to Aquaculture. A.A. Balkema Publishers.
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Figure legends
Fig. 1 Some anomalies caused by xenobiotics during the first cleavage: A: normal
cleavage; B: slightly asynchronous cell cycles of the 2 blastomeres; C 2 unequal
blastomeres, incomplete cleavage; D: three blastomeres, of different sizes. (Y. Yokota et
al, 2000. p. 84) Many of the embryos in experimental groups with BPA concentration
equal to or greater than 3uM have morphologies resembling B-D at the 2-cell stage.
Effects most prominent in groups with concentration greater than 5 uM.
Fig. 2. Control and experimental zygotes fixed in Carnoys and stained with acetocarmine
at 35 minutes after fertilization.
The experimental group is exposed to 7uM of BPA
Note the similarities between the two zygotes, and the fact that chromosomes were not
readily visible.
Fig. 3. Control and experimental zygotes fixed in Carnoys and stained with acetocarmine
at 1.25 hours after fertilization. Not much difference can be discerned between the two
groups. Chromosomes scattered near the middle of the zygote in both groups.
Fig 4. Control and experimental zygotes 2 hours after fertilization. Note the lining up of
the chromosomes in the control group, and the scattering of the chromosomes in the
experimental sample.
Fig 5. Control and experimental zygotes 2.25 hours after fertilization. Note the cleavage
furrow in the control group. The control group also has two sets of chromosomes. The
chromosomes in the experimental sample is still scattered.
Fig 6. Control and experimental zygotes 2.5 hours after fertilization. The control sample
pictured is almost ready to enter the 4-cell stage—the two sets of chromosomes are lined
up to divide again. The experimental, in contrast, dwells in the 1-cell stage. Its
chromosomes are still scattered and not lined up, showing that the cell is still at prophase.
Fig. 1
Fig. 2
Control
Experimental
Fig. 3
Control
Experimental
Fig. 4
Control
Experimental
Fig. 5
Control
Experimental
Fig. 6
Contro
Experimental