1. Figure 4: Protocol for Growth Assay.
The 96 well plate was placed into a
microplate reader and cells were
analyzed to capture the Optical Density
(OD) that was used to determine the
Inhibitory Concentration (IC) values.
Figure 5: Protocol for Pinning
Assay. The cells that were pinned
on the plated were let to grow for
three days and a Kodiak imager
was used to photograph the plates.
Figure 6: Protocol for Deletion Assay. Yeast Cells are grown in
liquid culture and treated (-URA Media, DMSO, Phenol)
or Untreated (Control: -URA Media and DMSO) for 17 hrs. Cells are
then washed, diluted and plated onto –Ura (viability) and –His
(recombinants) plates, grown for three days. Recombination
frequencies are determined: # of colonies -His Plates/ # of colonies -
URA plates. If frequency of Recombination is > than 2 fold compared
to untreated cells, this would indicate that DNA breaks occurred.
Growth Assay
Pinning Assay
Apoptosis Assay
Figure 7: Protocol and detection for Apoptosis Assay . Flow cytometry was used to analyze the cells after
incubation to see the percent of cells that had undergone apoptosis. Tee substrate is L-Asp(2)-rhodamine 110 (D2R)
that when cleaved by caspases fluoresces
Conclusions/Discussion
●As for the Apoptosis Assay, the results, though indicating that there is no significant
difference in induced apoptosis between the control and the treated yeasts, are
inconclusive due to the unforeseen fluorescence from the positive control assays.
●For the Deletion Assay, the pilot assay has given us an idea of the dilutions to use for
when we run the whole assay, results for phenoxyphenol show a potential two fold
increase in deletion formation . Because of time restrains, the deletion assays for
butylparaben, phenoxyphenol and trifluoromethyl phenol could not be conducted in
time.
●Based on our pinning assay, the IC 20 concentration of butylparaben possibly
encourages DNA damage in mutants that are defective in recognizing bulky DNA
lesions and maintaining chromatin structures. The removal of genes involved in double
strand break repair causes the yeast cells to be more resistant as the removal of those
repair genes allows the strain to be able to withstand DNA damage from butylparaben. It
seems that butylparaben causes damage that allows growth defects if processed by DSB
break protein or the lack thereof.
● Phenoxyphenol severely affected mutant strains that lacked genes involved in
repairing UV damage, or bulky DNA lesions. It possibly implies that phenoxyphenol
causes these types of DNA damage. However, among the resistant strains, many of the
strains survived with a lack of a nucleotide excision repair system. These genes are
involved in detecting UV light damage and bulky DNA lesions but their absences may
cause these damages to go unnoticed in DNA repair. Resistance may be due to other
repair systems bypassing these damages. However, the sensitive strains lack genes in
processing this damage. The process for removing these lesions are missing and cause
the mutants to be sensitive compared to the WT strain. This makes phenoxyphenol an
interesting compound to look at because the possible DNA damage it causes may be
dealt by a combination of repair pathways.
Cytotoxicity of Butylparaben, Phenoxyphenol and Trifluororomethyl
Phenol on Saccharomyces cerevisiae
Prisca Diala (‘18), Katiannah Moise (‘18), Dr. Cristina Negritto and Dr. Cynthia Selassie
Department of Molecular Biology, Pomona College
Summer of 2015
Abstract ResultsMaterials & Methods
Phenolic compounds are incorporated as antioxidants in the development of
cosmetics, plastics and processed foods. While previous research supports the
notion that the antioxidant nature of the phenols may function to suppress the
genotoxic effects of free radicals that may evolve as a result of oxidative reactions,
recent studies suggests that many of these same phenolic compounds can become
free radicals, causing oxidative stress themselves. This oxidative stress can induce
double strand breaks or the formation of DNA adducts, leading to mutations which
could result in these compounds being potential carcinogens. In the present study,
we focused on evaluating the cytotoxicity of butylparaben, 4-phenoxyphenol, and
4-trifluoromethyl phenol, three phenols that are commonly used in cosmetics. We
first determined the inhibitory concentration (IC) values at which these phenol
compounds may kill 20, 50 and 80 percent of yeast cells. We then screened
different null mutant strains of yeast cells for growth defects in the presence of
these phenols using a Pinning Assay. The strains screened all have one DNA
repair gene deleted and we observed that some of the strains were either resistant
or sensitive to the varying IC values of the phenols that they were exposed to. It is
possible that phenoxyphenol may cause significant DNA damage since mutants
that are defective in excising adducts and other types of bulky DNA damage were
sensitive. suggesting that they may not cause breaks but connect DNA together.
Butylparaben, it is suggested, may cause oxidative type damage since the screen
identified strains defective in base excision repair. To better understand the growth
defects, further experiments were done to determine if apoptosis is induced in
response to the phenols. However the results were inconclusive and future
experiments will focus on determining if infact there are incidences of apoptosis
invoked by the phenols. Deletion assays were done on strains to determine whether
these phenols cause double strand breaks in treated yeast strains. We are at the
preliminary stages of setting up this experiment and figuring out the right dilutions
of yeast cell cultures to use. Until we finalize these steps then will we be able to
reach any type of conclusion.
Acknowledgments
We would like to thank Pomona College for providing us with the funding to
conduct this research. In addition, we would like to extend our sincere
gratitude to Dr. Cristina Negritto for allowing us to work in her lab and
providing us with the equipment and guidance to effectively conduct our
research. Likewise, we also extend our sincere gratitude to Dr. Cynthia
Selassie for assisting us in the understanding of the structures of the various
phenolic compounds we worked with. Lastly, we would like to thank Sarai
Santos (‘16) for her input in the early stages of the experiment and Maria
Arciniega (‘16), Nancy Zhu (‘16) and Zoe Zhou (‘18) for their much
appreciated help throughout the whole process.
Introduction
Figure 8: Determination of IC values for phenolic compounds used in this study: A Kill Curve was
determined for each phenolic compound. Yeast cells were grown in the absence or presence of different
concentrations of phenols and the normalized OD vs. the log of [phenol] was plotted. The linear portion of the
curve is used to determine the IC20, IC50 and IC80values.
Figure 10: Phenol induced apoptosis in X70 yeast cells. Pictured above are histograms indicating fluorescence
of 5000 cells. Cells that have undergone apoptosis release the enzyme caspase that cleaves D2R, which yields a
fluorescent product. In the graphs, the region where the expected fluorescent cells would be are indicated with a
brace symbol (green for butylparaben and pink for phenoxyphenol and trifluoromethyl phenol.).The histograms
indicate that there is no significant difference in the number of apoptotic cells between the treated and untreated
cells.
Future Work
● We would like to conduct more Pinning Assays on trifluoromethyl phenol
because the plates we created did not grow.
● Further experiments are desirable for reaching a conclusive result in the
Apoptosis Assay.
● Additionally, we would like to test whether apoptosis induced damage by the
phenols would increase if the cells are treated with the phenols for 17hrs
rather than the 4hrs that they are being treated at now.
● For the deletion assay it would be excellent to conduct a more conclusive run
for IC20 and 50 values of butyl paraben, phenoxyphenol and trifluoromethyl
phenol. Hopefully we would be able to see at least two fold increase in
mutation rates among the phenols.
Incubate at 30°C for 17 hours
Incubate 17hrs
at 30°C
SC media,
X70 cells
(2x106cells/ml),
Phenolic compound,
1% or 2% DMSO
(totaling of 1000ul)
SC media,
X70 cells Pipette 200ul into 96
well plate
10ul of yeast cells were
pipetted into 190ul of fresh SC
mediaGrown to
saturation at
30·°C for 2
days.
Each column
contained a different
yeast strain and each
row contained a 1:10,
1:100, 1:1000 or
1:10000 dilutions of
the cell solution
Phenol
compound stock
or DMSO
Plates were created containing
SC media and either DMSO or
a phenolic compound
Deletion yeast
strains, SC media
Floating pin
replicator used to
dip into the 96 well
plate, pick up cells,
and then pinned
onto the plates.
● Phenolic compounds are commonly used as antioxidants in the industrial
synthesis of many consumer products like processed food and cosmetics.
● Previous experiments in the Negritto/Selassie lab have focused on the
cytotoxicity of phenols like BHT and BHA, which are mainly found in food
preservatives.
● The studies suggest that phenol compounds can indeed induce DNA breakage
in Saccharomyces cerevisiae cells.
● Our goal is to evaluate the cytotoxicity of butylparaben, phenoxyphenol and
trifluoromethyl phenol, three phenols which are found in cosmetics.
● To do so, we will conduct assays to first determine the IC values at which
these phenols induce DNA damage in yeast (Figure 1). Upon determining the
IC values, we will screen for growth defects in different DNA repair mutant
strains and using flow cytometry, determine whether the phenols induce
apoptosis in the cells (Figure 2). In addition, we will attempt to conduct a
Deletion Assay to determine if DNA breaks are caused by the introduction of
phenol compounds to yeast strains(Figure 3).
Figure 1: Mechanism for calculating IC
values. With increasing concentrations of
phenolic compounds, cellular growth
should be inhibited. The linear range of the
curve is used to determine the IC20, IC50,
and IC80 values for the specific phenol
compound.
Figure 3: DEL Assay. Yeast Strains X70,
X71 and X90 have a URA3 gene inserted
the HIS3 gene disrupting it DNA breaks
would cause the URA3 gene to be excised
and through homologous recombination,
the HIS3 gene will become functional.
The frequencies of recombination are used
to quantify the fold increase of deletion
formation between untreated and treated
strains.
Figure 2: Diagram of how flow cytometry
works. Yeast cells treated with an appropriate
substrate to detect apoptotic cells by fluorescence
(see figure 5) are in suspension and are taken up
by a capillary tube. As these cells flow through a
laser will cause apoptotic cells to fluoresce and a
detector will determine the number of cells that
have gone through and how many were
fluorescent. (Figure taken from abcam.com).
Yeas
t Cell
D2
R
D2R
Yeas
t Cell
D2
R
D2R
Yeas
t Cell
D2
R
D2R
Apoptotic
Cell
Normal Cell
Yeas
t Cell
D2
R
D2
R
Incubate for
4 hrs.
Transfer to disposable
10 ml tubes and spun
down. Discard media
resuspend in 1.5 ml of
PBS
X70, X71, &
X90 yeast
strains are
incubated in
SC media for
17 hrs.
Treated sample:
5x106 cells/ml +
phenol + DMSO
Cells are transferred to
eppendorf tubes and
washed 2X with PBS
Untreated
sample:5x106
cells/ml +
DMSO
Incubate in
the dark at
37°C for 30
min. Run thru
Flow
cytometer
D
2
R
D
2
R
1x105
cells/ml
treated with
D2R
IC20 IC50 IC80
0.02410
1 0.038465 0.061391
mM mM mM
IC20 IC50 IC80
1.011037 1.255611 1.559349
mM mM mM
IC20 IC50 IC80
0.165495 0.200154 0.242073
mM mM mM
Figure 9: Pinning assay as a measure of cell growth. Cell growth among mutant strains defective with one
or more class of DNA repair damage ( base repair, excision repair, etc) treated with phenols and dmso
compared to the wild type yeast strain determined growth/resistance and non-growth/non-resistance. Cell
growth was quantified using ImageQuantTL software based on pixelated intensity.
Red- Resistant Compared to WT
Blue- Sensitive Compared to WT
DMSO 8x12 Butyl Paraben 8x12
DMSO 8x11 Phenoxyphenol 8x11
DMSO 8x11 Butyl Paraben 8x11 DMSO 8x12 Butyl Paraben 8x12
DMSO 8x12 Phenoxyphenol 8 x12
Left to Right: Mutant Strains 8x12
rad3-R660C; rad3-G595R; rad1; rad3-A596P;
dnl4; hdf1; sir3,sir4; sir2,sir3; sir3; ogg1; apn1;
WT(1)
Left to Right: Mutant Strains 8x11
First Row: sae2; rad9; rad52; rad4; rad55; rad59;
rad27; mus81; srs2; sgs1; WT (1)
Second Row: WT (2); WT (3); pol3-1; rmr3; htz1;
rad3,rad4; rad2; rad14; rad4 (1); rad4 (2), WT(1)
-HIS
Untreated
-URA
Untreated/
Treated
-HIS Treated
Put 1 ml of
treated/untreated cells
into 4x 220 ul aliquots
into micro well plate
respectively
Wash
Cells of
phenol
and/or
DMSO
Treated Assay
Untreated Stock
Plate 100ul of each cell
solution onto appropriate
plate based on phenol and
dilution and let grow for 3
days-URA Media
-1%,2%,3% DMSO
-DMSO + Selected
phenol
-Incubated Yeast
Strain (17 hrs.)
-URA Media
-1%,2%,3% DMSO
-Incubated Yeast
Strain (17 hrs.)
Deletion Assay