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Endocrine Disruptors in Yeast (Saccharomyces cerevisiae)
Principal Investigator: Madeline Strong, Undergraduate student,
Department of Biology, Tennessee Technological University, Cookeville,
TN 38501
October 30, 2007
Table of Contents
Project Summary…Page 3
Key Words…Page 3
Introduction… Page 4
Methods and Materials…Page 6
Expected Results…Page 9
Project Timeline…Page 10
Literature Cited…Page 11
Budget…Page 14
Project Summary
I studied endocrine disruption in the budding yeast cell cycle. The objective of
this experiment was to determine exactly how endocrine disruptors affect cell cycle. It is
known that they affect cells in some way, hence mutations. It is my hypothesis that
endocrine disruptors stop the cell mid- cycle, eventually killing the cell and in prolonged
exposure, killing the organism. To test this theory, I have grown and incubated three
separate colonies of Saccharomyces cerevisiae, common baker’s yeast. The yeast will be
stained and then exposed to different endocrine disruptors. Microscopy will be used to
determine the effects of endocrine disruptors on these cells. I expect that the cells will
stop in their cycles, which will be viewable via the nucleus under the microscope. I also
expect the cells to die under prolonged exposure to the endocrine disruptors. By studying
the budding yeast cell cycle, a model eukaryotic cell, endocrinologists can better
understand the effects of endocrine disruptors on higher order eukaryotic organisms, such
as vertebrates.
Key Words: Cell cycle, cell mutation, endocrine disruption, Saccharomyces cerevisiae,
yeast
Introduction
Background of Literature Review
Endocrine disruption has been a topic of concern for endocrinologists for quite
some time. Endocrine disruptors (EDs) are chemicals that have the ability to interfere
with hormone signaling pathways (Landrigan, Garg, & Droller, 2003), and many of these
substances have been linked to developmental, reproductive, and other health issues
(Spearow, Doemeny, Sera, Leffler, & Barkley, 2004). Effects of EDs have been observed
in many contaminated ecosystems and, to a limited extent, in humans. For example, two
studies in 2003 (one in Japan and one in the U.S.) linked exposure to EDs in utero to
cancers in children (Mori, Komiyama, Adachi, Sakurai, Nishimura, Takashima, &
Todaka, 2003), and a 2004 study linked EDs exposure to testicular cancer (Hardell,
Bavel, Lindstrom, Carlberg, Eriksson, Dreifaldt, Starkhaamer, Hallquist, & Kolmer,
2004). Some EDs are produced for specific purposes and others are by-products of other
chemicals or manufacturing. Just a few examples of endocrine disruptors are pesticides,
products associated with plastics, pharmaceuticals, household cleaning supplies, and
industrial chemicals. Based on this information we have to ask ourselves, how do
endocrine disruptors affect so many different types of organisms? In theory, the
endocrine disruptors stop the cell mid-cycle, which kills the cell and eventually the whole
organism.
We have used the budding yeast cell cycle, Saccharomyces cerevisiae, a simple
dynamic model that demonstrates that the cell cycle is extremely stable and robust for its
function (Li, Long, Lu, Quyang, & Tang, 2004). About 800 genes are involved in this
cell cycle (Simon, Barnett, Hannett, Harbison, Rinaldi, Volkert, Wyrick, Zetlinger,
Gifford, Jaakola, & Young, 2001). The process consists of four phases: G1 (the cell
grows and commits to division), S (DNA is synthesized and chromosomes are
replicated), G2 (a gap between S and M), and M (the chromosomes are separated and the
cell is divided in two). After the M phase, the cell enters G1 once again and completes
the cycle (Botchan, 1996). The cell cycle is regulated by cyclins, inhibitors, degraders,
transcription factors, and checkpoints. Proteolysis, the digestion of proteins by cellular
enzymes, is vital to the cell cycle because it initiates DNA replication, the separation of
sister chromatids, exit from mitosis, and is responsible for the degradation of cyclins at
different stages of the cycle (Baumer, Braus, & Irniger, 2000). When proteolysis is
inhibited, it causes a mutation that stops cells in their cycles (Thornton, Chen, Cross,
Tyson, & Toczyski, 2004).
Statement of Problem
Endocrine disruptors affect so many different organisms, so endocrine disruptors
must work in a common way. All organisms have cells, and all of those cells are
constantly going through their cycles. So how do endocrine disruptors affect the cell
cycle?
Objective/ Null Hypothesis Statements
The objective of this research is to determine the effects of endocrine disruptors
on the cell cycle. The budding yeast cell cycle was chosen because it is a simple model of
the eukaryotic cell. The null hypothesis is that the endocrine disruptors will have no
effect on the yeast cell cycle.
Methods and Materials
Statistical Approach
I used strains of Saccharomyces cerevisiae grown at room temperature, in reduced
light and in open Ziplock bags to keep the yeast cultures from drying out too quickly.
They were supplemented with 1% sucrose (sugar) solution.
To estimate the number of yeast cells in a culture, place 1 ml of sterile water into
a tube. Take a sterile toothpick and suspend a small amount of yeast, about the size of a
pinhead into the water. Swirl the solution thoroughly. Dilute 1 ml of your solution and
swirl it. Then put 1 ml of just sterile water into another tube. If the tube with cells is
turbid just to the limit of visibility, then it contains nearly 1 x 106 cells/ml. If it is more
turbid than the limit of visibility, then add more sterile water. If it is not turbid, add .1
volumes of the original suspension until it is. This method is from the KSU Physics Lab
Manual. After I have estimated the number of cells, I will add the endocrine disruptor
solutions to the colonies. To observe the effects, use a low power objective (10x)
microscope. Put a drop of water on the microscope slide and transfer a small amount of
yeast to the water with a sterile toothpick and stir a little. Put a coverslip over the slide.
Add 1 drop methylene blue solution (to observe the nucleus.)
Expected Results and Benefits
I expect the cells to stop in their cycle, which will be viewable under the
microscope. This research is beneficial to the field because the robust cell cycle of the
budding yeast is the same cell cycle of every eukaryotic organism, including humans. If
the endocrine disruptors affect the yeast cell cycle in this way, then it is likely that they
will affect other eukaryotic cell cycles, including humans, the same way. Furthermore,
research in the area of cancer prevention would drastically improve. Birnbaum and
Fenton (2003) suggest that polychlorinated biphenyl congeners play a role in the
aetiology of testicular cancer if exposed when in utero. With a continued research into
exactly how endocrine disruptors affect the body, endocrinologists could find another
route of prevention.
Budget
Personnel
Primary Investigator
Equipment
Thermometer
$20
Materials and Supplies
Fish Tank
$20
Test Tubes
$20
Methylene blue solution
$7
Plates
$10
Ziplock Bags
$3
Toothpicks
$1
Total
$81