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Figure 3: This sigmoidal model, based on our mortality data after applying logistic
regression, was used to calculate the most precise LC50. The relationship depicted is
between gaseous toluene exposure in ppm and probability of fly death.
(ppm)
Determining the Correlation between Toluene Exposure on Drosophila melanogaster
and the Resulting Toxicity Effects of Toluene on Fly Offspring Morphology
Luke Hebert1, Gina Duong1, Cecilia Dao1, Katie Fisher1, Heidi Reinhardt1, Quy Lam1, Vanessa Trivino1, Elmer B. Ledesma2 and Rosemarie C. Rosell1. 1Department of Biology,
University of St. Thomas, Houston, TX 77006, 2Department of Chemistry and Physics, University of St. Thomas, Houston, TX 77006
Introduction
Toluene sources include petroleum refineries, automobile emissions, and household products like
paint and nail polish (4). It is classified as noncancerous but air toxic (1).
The average concentration of toluene in the air of U.S. urban areas is 2.66 x 10 -4 ppm, but workers
may be exposed to 50-200ppm, which may cause subtle changes in human neurological function (4, 5).
According to the U.S. Department of Health and Human Services, chronic toluene exposure correlates
to irregular fetal development in humans. Moreover, toluene negatively affects larval development
and fly emergence in Drosophila melanogaster (3).
Our ultimate goal is to determine the genetic correlation between toluene exposure and
developmental defects in a model organism D. melanogaster, the common fruit fly. We hypothesize
that the LC50 of toluene for fruit flies will be in a low ppm range and that toluene, when exposed to
female flies, affects the morphology and mortality of larval offspring.
LC50 determination:
The calculated LC50 for adult flies based on our logistic regression modeling is 279ppm (shown in Fig. 3
as a vertical line). While similar fruit fly mortality data was not readily available, mice have an LC50 of
440 ppm with shorter (24-hour) exposure (2).
More comparable data will yield a more accurate LC50. To obtain statistically comparable results, we will
conduct additional male and female trials at matching toluene exposure levels and collect offspring for
additional studies. We will also determine if the LC50 for males and females differs significantly.
Larval Development:
Only larvae of unexposed parents have been observed thus far (Figs. 4 and 5), however these controls
are yielding data that allow us to refine our methods of light and scanning electron microscopy so that
we will have comparable morphological data for the larvae with toluene-exposed parents.
We are currently collecting larvae from every combination of parents exposed at different
concentrations. Our progress has been slowed by equipment malfunctions and calibration problems in
our toluene delivery system but we are working to repair equipment and continue trials.
Acknowledgements
We gratefully acknowledge financial support from the U.S. Department of Education, Office of Post Secondary
Education, Hispanic-Serving Institutions, STEM Articulation Grant P031C110128, the Cullen-Smith Foundation, and the
University of St. Thomas Committee on Student Research for the financial support necessary to progress this study. We
appreciate the valuable advice of biostatistician Jack Follis and thank our past members for their essential
contributions. We also thank our advisors, Dr. Rosemarie Rosell and Dr. Elmer Ledesma, for their guidance over the
years.
Methods
Toluene exposed to
adults
Known levels of toluene exposed to
10-20 adults per trial for 48-hour
period.
System comprised of two flow
meters, an impinger, and gas
chamber (Fig. 1)
Light Microscopy
Photographs of 3rd instar
larvae were taken using an
inverted light microscope at
40X to 100X magnifications.
Larvae Preparation
1. 5% Glutaraldehyde fix
2. Ethanol washes
3. Critical point drying using CO2
Scanning Electron
Microscopy
Dried larvae were gold
sputter-coated and viewed
with a scanning electron
microscope.
Figure 1: Depicts airflow through our experimental setup comprised of two flow-meters, a toluene
impinger, and a gas chamber. Outside of this picture are two gas tanks supplying air and a ventilation
hood for gaseous waste output.
Figure 2: Relationship between gaseous toluene exposure in parts per million and
average mortality of flies exposed. Red points are averages of female % mortality
from replicate trials and blue points are averages of male % mortality. Toluene
exposure at each concentration was carried out in at least triplicates, with each
replicate trial containing about 10-20 flies.
Figure 4: Light microscope image of a live 3rd instar control larva from an air-
exposed female mated to an unexposed male at magnification 40X.
Figure 5: SEM image of a 3rd instar control larva from an air-exposed
female mated with an unexposed male after critical point drying and
sputter-coating.
Results and Discussion
[Toluene] vs. % Average Mortality
Literature Cited
1. ”2005 National-Scale Air Toxics Assessment.” Environmental Protection Agency. 1 January 2011. [cited November 8, 2014]. Available from
2. Material Safety Data Sheet: Toluene MSDS. ScienceLab.com: Chemicals & Laboratory Equipment.
3. Sinh MP, Reddy MMK, Mathur N, Saxena DK, Chowdhuri DK. 2009. Induction of hsp70, hsp60, hsp83, and hsp26 and oxidative stress markers in benzene, toluene, and
4. “Toluene.” Environmental Protection Agency. Technology Transfer Network Air Toxics Web Site, 27 July 2012. Web [cited October 12, 2013]. Available online at
5. Toxicological Profile for Toluene. Rep. Atlanta, Georgia: Agency for Toxic Substances and Disease Registry, 2000. Web. 6 Nov. 2013. Available online at
<<http://www.epa.gov/ttn/atw/nata2005/>>.
<http://www.hmdb.ca/system/metabolites/msds/000/032/826/original/HMDB34168.pdf?1358896073>.
xylene exposed Drosophila melanogaster: Role of ROS generation. Toxicol Appl Pharmacol. 235(2): 226-243.
<<http://www.epa.gov/ttn/atw/hlthef/toluene.html>>.
<<http://www.atsdr.cdc.gov/ToxProfiles/tp56.pdf>>.

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USTsymposium_FlyGroup_2015-VER7-lh

  • 1. Figure 3: This sigmoidal model, based on our mortality data after applying logistic regression, was used to calculate the most precise LC50. The relationship depicted is between gaseous toluene exposure in ppm and probability of fly death. (ppm) Determining the Correlation between Toluene Exposure on Drosophila melanogaster and the Resulting Toxicity Effects of Toluene on Fly Offspring Morphology Luke Hebert1, Gina Duong1, Cecilia Dao1, Katie Fisher1, Heidi Reinhardt1, Quy Lam1, Vanessa Trivino1, Elmer B. Ledesma2 and Rosemarie C. Rosell1. 1Department of Biology, University of St. Thomas, Houston, TX 77006, 2Department of Chemistry and Physics, University of St. Thomas, Houston, TX 77006 Introduction Toluene sources include petroleum refineries, automobile emissions, and household products like paint and nail polish (4). It is classified as noncancerous but air toxic (1). The average concentration of toluene in the air of U.S. urban areas is 2.66 x 10 -4 ppm, but workers may be exposed to 50-200ppm, which may cause subtle changes in human neurological function (4, 5). According to the U.S. Department of Health and Human Services, chronic toluene exposure correlates to irregular fetal development in humans. Moreover, toluene negatively affects larval development and fly emergence in Drosophila melanogaster (3). Our ultimate goal is to determine the genetic correlation between toluene exposure and developmental defects in a model organism D. melanogaster, the common fruit fly. We hypothesize that the LC50 of toluene for fruit flies will be in a low ppm range and that toluene, when exposed to female flies, affects the morphology and mortality of larval offspring. LC50 determination: The calculated LC50 for adult flies based on our logistic regression modeling is 279ppm (shown in Fig. 3 as a vertical line). While similar fruit fly mortality data was not readily available, mice have an LC50 of 440 ppm with shorter (24-hour) exposure (2). More comparable data will yield a more accurate LC50. To obtain statistically comparable results, we will conduct additional male and female trials at matching toluene exposure levels and collect offspring for additional studies. We will also determine if the LC50 for males and females differs significantly. Larval Development: Only larvae of unexposed parents have been observed thus far (Figs. 4 and 5), however these controls are yielding data that allow us to refine our methods of light and scanning electron microscopy so that we will have comparable morphological data for the larvae with toluene-exposed parents. We are currently collecting larvae from every combination of parents exposed at different concentrations. Our progress has been slowed by equipment malfunctions and calibration problems in our toluene delivery system but we are working to repair equipment and continue trials. Acknowledgements We gratefully acknowledge financial support from the U.S. Department of Education, Office of Post Secondary Education, Hispanic-Serving Institutions, STEM Articulation Grant P031C110128, the Cullen-Smith Foundation, and the University of St. Thomas Committee on Student Research for the financial support necessary to progress this study. We appreciate the valuable advice of biostatistician Jack Follis and thank our past members for their essential contributions. We also thank our advisors, Dr. Rosemarie Rosell and Dr. Elmer Ledesma, for their guidance over the years. Methods Toluene exposed to adults Known levels of toluene exposed to 10-20 adults per trial for 48-hour period. System comprised of two flow meters, an impinger, and gas chamber (Fig. 1) Light Microscopy Photographs of 3rd instar larvae were taken using an inverted light microscope at 40X to 100X magnifications. Larvae Preparation 1. 5% Glutaraldehyde fix 2. Ethanol washes 3. Critical point drying using CO2 Scanning Electron Microscopy Dried larvae were gold sputter-coated and viewed with a scanning electron microscope. Figure 1: Depicts airflow through our experimental setup comprised of two flow-meters, a toluene impinger, and a gas chamber. Outside of this picture are two gas tanks supplying air and a ventilation hood for gaseous waste output. Figure 2: Relationship between gaseous toluene exposure in parts per million and average mortality of flies exposed. Red points are averages of female % mortality from replicate trials and blue points are averages of male % mortality. Toluene exposure at each concentration was carried out in at least triplicates, with each replicate trial containing about 10-20 flies. Figure 4: Light microscope image of a live 3rd instar control larva from an air- exposed female mated to an unexposed male at magnification 40X. Figure 5: SEM image of a 3rd instar control larva from an air-exposed female mated with an unexposed male after critical point drying and sputter-coating. Results and Discussion [Toluene] vs. % Average Mortality Literature Cited 1. ”2005 National-Scale Air Toxics Assessment.” Environmental Protection Agency. 1 January 2011. [cited November 8, 2014]. Available from 2. Material Safety Data Sheet: Toluene MSDS. ScienceLab.com: Chemicals & Laboratory Equipment. 3. Sinh MP, Reddy MMK, Mathur N, Saxena DK, Chowdhuri DK. 2009. Induction of hsp70, hsp60, hsp83, and hsp26 and oxidative stress markers in benzene, toluene, and 4. “Toluene.” Environmental Protection Agency. Technology Transfer Network Air Toxics Web Site, 27 July 2012. Web [cited October 12, 2013]. Available online at 5. Toxicological Profile for Toluene. Rep. Atlanta, Georgia: Agency for Toxic Substances and Disease Registry, 2000. Web. 6 Nov. 2013. Available online at <<http://www.epa.gov/ttn/atw/nata2005/>>. <http://www.hmdb.ca/system/metabolites/msds/000/032/826/original/HMDB34168.pdf?1358896073>. xylene exposed Drosophila melanogaster: Role of ROS generation. Toxicol Appl Pharmacol. 235(2): 226-243. <<http://www.epa.gov/ttn/atw/hlthef/toluene.html>>. <<http://www.atsdr.cdc.gov/ToxProfiles/tp56.pdf>>.