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 SelassieDepartment of Molecular Biology, Pomona College

Summer of 2015

Abstract ResultsMaterials & MethodsPhenolic 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.

AcknowledgmentsWe 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 hoursIncubate 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

Phenolic compound (Treated)

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.

DMSO (untreated)

● 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).

Yeast Cell

D2

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D2

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Yeast Cell

D2

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D2R

D2

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Yeast Cell

D2

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Apoptotic Cell

Normal Cell

Yeast Cell

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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

D2

R

D2

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1x105 cells/ml treated with D2R

IC20 IC50 IC800.02410

1 0.038465 0.061391mM mM mM

IC20 IC50 IC801.011037 1.255611 1.559349mM mM mM

IC20 IC50 IC800.165495 0.200154 0.242073mM 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 WTBlue- 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 8x11First 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