This study compared the cytotoxicity and induction of genes encoding metabolic enzymes in cells exposed to different alkylated polycyclic aromatic hydrocarbons (PAHs). An SRB assay showed little cell death between exposures of 1uM naphthalene, 1uM dimethyl naphthalene, 1uM BAP, and the vehicle control, except for 5uM BAP. RT-PCR analysis found that 1uM BAP induced expression of CYP1A1 and CYP1B1 genes, but 1uM naphthalene and dimethyl naphthalene did not induce gene expression. Comparing responses to the parent naphthalene and dimethylated naphthalene,

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Comparative Induction of Genes Encoding Canonical Metabolic Enzymes by Alkylated Polycyclic Aromatic Hydrocarbons
J. Farve1,2, J. Wickliffe1,3, B. Simon1,3, A. Williams1,3
1 Emerging Scholars Environmental Health and Science Academy, 2 New Orleans Charter Science and Mathematics High School, 3 Department of Global Environmental Health
Sciences, Tulane University School of Public Health and Tropical Medicine
Materials and Methods
Introduction
Discussion and Conclusion
Abstract
Results
DMSO
1 uM BAP 5 uM BAP
1 uM NAPH
1 uM DMN medium
0
20
40
60
80
100
120
DMSO 1 uM BAP 5 uM BAP 1 uM NAPH 1 uM DMN medium
%ofDMSO Concentration of exposed chemicals
Acknowledgments
Figure 1. SRB Assay showing cell
viabilities to different exposures after
24 hours.
Figure 2. RTPCR Graph showing gene
expression during 40 cycles in BioRad
Thermal Cycler
Figure 3. CYP1A1
expression to cells exposed to
chemicals of interest after 24
hours
Figure 4. CYP1B1
expression to cells exposed
to chemicals of interest after
24 hours
Figure 5. expression of CYP1B1 in
vehicle and positive controls
Pervious research from the National Cancer Institute and the National Institute of Diabetes & Kidney Disease took note that kidney failure or chronic kidney diseases are increasing every year. Almost 10% of the American adult population
is affected and 90% of those cases are due to the exposures within our natural environment that is PAHs (Polycyclic Aromatic Hydrocarbons). PAHs can be produced from almost any combustion. For instance, volcanic eruptions, wildfires,
fossil fuels, waste products from vegetation…etc. Not all PAHs are harmful, but most of them can be very toxic especially when ingested and metabolized. Recently, the Gulf Region experienced an oil spill that severely damaged local
environments which made several communities vulnerable to many toxic PAHs. Locally the PAHs that we take in comes from our seafood, therefore, making this study so vital. Those toxic PAHs that’s within the seafood eventually gets
digested and later metabolizes, making the toxicants worsen. Even the smallest amount of those toxic PAHs overtime prove to have an effect on the human body.
Cell Culture:
RPTEC/TERT1 cells, purchased from Evercyte Laboratories, were cultured in
75 milliliter tissue culture flasks. We split 70-80% confluent flasks at a 1:2
ratio once a week.
SRB Cytotoxicity Assay:
SRB (Sulforhodamine B) is a stain that binds to proteins present in cells, and
is a tool for measuring cell density, therefore, we can compare cell viability
to the different exposures. We plated 100,000 cells per milliliter in 96 well
plates and exposed them to 1uM Naphthalene, 1uM Dimethyl Naphthalene,
1uM BAP, 5uM BAP, and the vehicle control 1uM of Dimethyl sulfoxide
(DMSO) that were exposed for 24 hours. Cells were fixed using
Trichloroacetic acid (TCA) and stained with SRB for 30 minutes. Then the
plate was lysed with Tris base solution. Later the plate was read at 488- 589
nm using the Tecan infinite M200 Pro featuring the icontrol software.
RTPCR:
1.RPTEC cells were confluent in 12 well plate and were exposed to the same
chemical described above for 24 hours in 3 groups. RNA was extracted
from the cells using the QIAshredder and RN easy kit. Then the samples’
concentrations and qualities were measured using the Nanodrop 2000c
spectrophotometer. Later the samples were diluted with RNase free water
to produce 1ug of cDNA in 20 uL total solution.
2.cDNA was synthesized using BioRad iScript reaction mix and reverse
transcriptase. Then the samples were placed in the BioRad C1000 Thermal
Cycler featuring CFX96 Real-Time PCR Detection System.
3.Next the CY1A1 and CY1B1 gene expression were measured using CY1A1
and CY1B1 primers applied by the TaqMan Gene Expression Assays. The
samples were later compared to reference genes GAdBp and ACTB for
quality assurance.
I want to thank my family for being my biggest fans
and being my support group this summer. Next, I
want to thank my lovely mentor Ms. Addie Williams
who was a definite help to me this summer! Thank
you Dr. Simon and Dr. Wickliffe for giving me the
opportunity in your lab for my emerging scholar’s
project. And last but never least, thank you Mrs.
Perrault for believing in me. This work was
supported by the Gulf Region Health Outreach
Program (GRHOP) which is funded from the
Deepwater Horizon Medical Benefits Class Action
Settlement approved by the U.S. District Court in
New Orleans on January 11, 2013.
Shown in the SRB graph (Figure 1.) not much cell death was displayed except for the cells that were exposed to 5uM of
BAP. In the RTPCR graph (Figure 2.) gene expression was shown in cycle 20. Not much of a difference was shown between
the Naphthalene and the Dimethyl Naphthalene. CYP1B1 shows expression when exposed to the positive control BAP
verses the vehicle control DMSO.
Based off the data that is displayed, SRB Assay didn’t
show significant decrease in cell viability except for
the concentration of 5uM of BAP.
1 uM BAP induces gene expression of CYP1A1 and
CYP1B1. However, exposure to 1uM of Naphthalene
and the Dimethyl Naphthalene didn’t show gene
expression. These results are valuable because the
quality was proven to be excellent due to the two
controls that didn’t include any samples nor reverse
transcriptase.
Comparing the parent compound Naphthalene to the
Dimethylated Naphthalene, cell viabilities and
CYP1A1 and CYP1B1 gene expression showed little
variation.
Polycyclic Aromatic Hydrocarbons, or PAHs, can be produced from incomplete combustion. Not all PAHs are harmful, but most of them can be very toxic especially when ingested and metabolized. The purpose of this experiment is to
compare degrees of cytotoxicity between a well-defined PAH, Benzo[a]pyrene (BAP), in two different concentrations, as well as the simplest PAH, Naphthalene, and a similar variant, Dimethyl Naphthalene and exposing those
concentrations to RPTECs. We found that after a 24 hour exposure, cell viability did not show a significant decrease between the chemicals. 1 uM BAP induces gene expression of CYP1A1 and CYP1B1 genes which encode metabolic
enzymes while exposure to the vehicle control, DMSO, does not exhibit e3xpression. However, exposure to 1uM of Naphthalene and the Dimethyl Naphthalene didn’t show gene expression. We conclude that cells exposed to the parent
compound Naphthalene versus the alkylated dimethyl naphthalene do not significantly cell viability and CYP1A1 and CYP1B1 expression showed little variation.