Title: Effect of Aflatoxins on T and B Lymphocyte Function
Names: XXXXXXXXXXXXXXXX
Abstract:
Aflatoxins are metabolites of mold commonly found in food supplies in areas with a
warmer tropical climate conducive to mold growth. These toxins are most commonly found in
foods such as nuts and grains, particularly in developing countries where food storage is less
regulated. Because of their prevalence, aflatoxins have already been studied in order to find their
impact on human health and have been identified as carcinogens most commonly responsible for
liver cancer. Animal studies have suggested that these toxins may also have a negative impact on
immune function- causing immune cell death and changes in protein production- but the effects
on human cells have not been thoroughly studied. In particular the effect of these toxins on the
functioning of T and B cells, which are responsible for identifying and killing pathogens, is
limited. This study will investigate the potential adverse effects of these aflatoxins on immune
cells.
Personal Statement:
There have been two areas of academics that have captured my fascination since middle
school- biology and chemistry. So far at Elon, however, my interests have been expanding. My
time at Elon has allowed me to explore areas that I never would have imagined. My path has not
narrowed, but rather has become more branched and winding than ever. For instance, through
my Global Experience class as well as my time in the Periclean Scholars and Campus Kitchen
programs, I have developed a strong interest in poverty and development studies. I want to
further understand the mechanisms behind the perpetuation of poverty as well as ways to
alleviate it. This interest has led me to others in areas such as nutrition, political science, public
health, sustainable agriculture, and even Arabic.
When developing my research project last spring, I never expected to reach a
compromise between my rapidly expanding areas of interest and my distinct biological and
chemical interests. I decided to focus on something that particularly excited me about
biochemistry research: the immune system, where chemical signals are not only used to
communicate between cells, but to identify and destroy foreign pathogens. I am passionate about
understanding not only how it works, but what situations cause it not to work. As I looked
further into what I wanted to study, I found information about aflatoxins, which have been
shown to be capable of entering the body and preventing our immune systems from functioning
correctly. But how this occurs has not yet been discovered. Both my interest in immune function
and my passion for finding chemical explanations for biological occurrences drew me to this
project. As I researched more I found that it connected to my broader interests as well. These
aflatoxins are most commonly found in developing countries, where food storage is less
regulated and crops such as corn and grains are exposed to mold. The suppressed immune
function caused by aflatoxins could be partially responsible for the prevalence of many of the
infectious diseases in that area. Because of this, my project has not only allowed me to
investigate the chemical interactions of the immune systems, but the larger systems behind the
perpetuation of poverty as well.
When I look at where I will be in five years, I am not sure exactly which path I will take.
I know I am interested in the medical field, either discovering the mechanisms behind diseases
that plague impoverished areas or working to stop them, but I haven’t yet discovered what area is
the best fit. This project, however, has already helped me see the possibilities for a future career
that can combine my multifaceted interests. Receiving the Lumen prize would allow me to
explore this project much further, allowing me to gain even more of an understanding of where
my passions lie and where they can take me. I will be able to learn more advanced techniques
inside of the lab, while also learning how to be a part of a wider community of scholars who
share my passions. The prize would allow me to increase the quality and quantity of the
experiments that I will perform, through the use of materials and technology of a higher quality
and wider variety than those that I would be able to use otherwise. It will allow me to travel to
share my findings, helping me to find my place in the scientific community. Overall, the product
of my research and my personal growth that will result from it will be greatly supported by the
Lumen Prize.
Part II: Project Description
Focus:
Aflatoxins are secondary metabolites of two common mold species, Aspergillus flavus and
Aspergillus parasiticus, known for the contamination of animal feeds as well as staple foods
such as groundnuts, corn, rice, spices and seeds (1,2). This contamination is common in animal
feed as well as food supplies in developing countries, where food-production technology is not
developed enough to avoid the conditions conducive to mold growth (2). It is estimated that
approximately 4.5 billion people living in developing countries are regularly exposed to this
toxin in uncontrolled concentrations (2). In 2010, a study conducted in Texas found that onequarter of the participants had exposures above the daily limit set by the FDA (11). This shows
that even in more developed areas where aflatoxin limits are set, significant exposure still occurs.
Aflatoxins have been found to be a significant contributor to many acute and chronic health
issues, including liver cancer, nutritional interference, and immunosuppression. A study in 2010
estimated that between 4.6% and 28.2% of liver cancer cases worldwide can be attributed to
aflatoxin exposure (9). Wide-spread estimates of the effects of aflatoxins on other global health
issues have not been as thoroughly investigated.
While the role of aflatoxins as a carcinogen in liver cancer has been well-studied, more
recently it has been investigated as an immunosuppressant in clinical studies. In a study of
Gambian children, those whose blood samples yielded a positive result for aflatoxin exposure
showed decreased concentrations of immune proteins in saliva (4). Other studies have found that
aflatoxins have a negative impact on cell-mediated immunity. A study on Ghanians found that
there was significant aflatoxin exposure in all of their participants. There was a significant
decrease in activation of multiple forms of immune cells, including T-cells, B-cells, and
macrophages in the participants with “high” aflatoxin levels (greater than 0.9068 pmol/mg) in
comparison to those with “low” aflatoxin levels (1). Aflatoxin exposure has also been shown to
have a negative effect on the immune function in humans infected with the Human
Immunodeficiency Virus and has been shown to speed up the progression of the disease (5,2).
Though these clinical studies are useful in identifying the symptoms and diseases correlative
with increased aflatoxin exposure, how these aflatoxins interact with our cells and body systems
in order to cause these effects cannot be identified. This proposal aims to study the effect of
aflatoxin on immune cell function in order to more comprehensively understand the mechanism
underlying its immunosuppressive effects.
The current research on the effect of aflatoxins on specific immune cells is mainly focused
on the functioning of macrophages, which have been shown to be impaired with increasing
concentration of aflatoxin (3,6). However, the effects of aflatoxins on other immune cell groups
important in the recognition and elimination of pathogens, such as T-lymphocytes and Blymphocytes, have not been thoroughly studied. T-cells are responsible for signaling other major
cells of the immune system in order to initiate the immune response and can also directly kill
infected cells. These cells are also important for the development of immunological memory,
which allows for a more rapid attack of pathogens in recurrent exposure. Mice treated with
increasing concentrations of aflatoxin were found to have reduced populations of helper-T cells
and B cells as well as reduced antibody responses (10). The proliferation of T-cells is primarily
regulated by the production of the IL-2 protein, which was also shown to be present in lower
concentrations in the mice treated with aflatoxin. A study performed on plated, human leukemic
T-cells focused on the effect of aflatoxins on the production of IL-2. A study found that when
stimulated to produce IL-2, the T-cells that were exposed to aflatoxin had lower levels of protein
as a result of an impaired production mechanism (7). Therefore, the research on the immune
responses of mice suggests that aflatoxins have an adverse effect on T-cell function (2, 7). The
aforementioned study performed on blood samples of Ghanaian individuals also suggested that
aflatoxins reduced the number of activated T and B cells in the blood samples of their
participants, yet the reasons for these decreases are not yet known (1). Other than these studies,
the research surrounding the effects of aflatoxins on immune cells is limited.
The goal of this research will be to gain further understanding of the effect of aflatoxins on
the cells of the human immune system. Based on studies performed on the immunological
responses of both animals and humans, it is expected that aflatoxins negatively impact the T and
B lymphocyte numbers either by inducing cell death and/or reducing cell growth. As a result, we
would also expect the proteins involved in replication to be down-regulated or the proteins
responsible for cell death to be up-regulated. To test these hypotheses, human T or B cells will
be cultured with or without aflatoxin. The growth of T and B lymphocytes will first be
investigated through cell counts as well as assays of signaling proteins involved in the regulation
of the cell cycle. Cell death will also be studied through the use of viability tests and flow
cytometry. If the results indicate that cell death is occurring, further study of apoptotic proteins,
which signal for programmed cell death, will be used to identify the mechanism behind this
occurrence. In the case of either impaired growth or increased death signaling, antioxidant
compounds can be introduced to cells exposed to aflatoxin in order to see if the harmful effects
would be reduced as seen in the studies involving liver cancer (8).
Regardless of whether growth or death is occurring, aflatoxins may be modulating immune
cell function through dysregulation of enzymes used in signaling, activation, and response to
pathogens. If cell growth and death are not significantly affected, the immune response of the
cells can be assessed. These responses would be studied by eliciting an immune response after
exposing the cells to aflatoxin. Production of cell-signaling and activation proteins involved in
identifying and responding to pathogens will then be analyzed through the use of assays and
compared to the protein production in cells not exposed to the toxin. These results will be used to
better understand the effects of aflatoxins on these cells.
Proposed experiences:
Research for this project will continue during summer of 2013 through the Summer
Undergraduate Research Experiences program. During this time, I will be taught various
analytical techniques including cell culturing, as well as various protein assays and blotting
techniques. Because of the amount of time that this experience offers, I will have the opportunity
to perform many tests on the effects of aflatoxin on cell viability and protein production in order
to establish a basis of understanding of those effects. This experience will also offer many
opportunities for presentation, including informal departmental presentations as well as formal
presentations at the end of the summer and in the fall.
I will continue to further develop the project over the next two years at Elon. The
techniques learned during the summer of 2013 will allow me to further investigate the aflatoxin
effects and further develop my skills in the lab. I plan to take advantage of other opportunities to
present my research both at Elon, through programs like the Student Undergraduate Research
Forum, and at regional or national conferences such as the American Society of Cell Biology
Annual Meeting.
To aid in the development of my knowledge and skills, coursework will be selected that
provides further insight into my project. I will take Biochemistry as well as Quantitative
Chemical Analysis, Human Anatomy, and Microbiology all of which will help me understand
processes involved in my research and will build on my laboratory skills. Coursework such as
Public Health, Epidemiology, and Nutrition will help me to further understand the bigger picture
that my project fits in to.
During the winter of 2014 I also hope to participate in the Public Health Study abroad
program to northern India in order to work with the Community Rural Health Project. During
this experience, I will be able to work with women in the area, as they train to become
community health workers, as well as professionals in the field of Public Health. This will allow
me to understand the areas that are affected by the toxin that I am studying, while also gaining a
better understanding of other health issues in the region.
Proposed products:
The products of this project will be mainly in the area of scholarly work intended for
presentation and publication. The findings that I make over the next two years of my research
will be presented, starting with departmental and program presentations for SURE 2013. I hope
to also present in the Student Undergraduate Research Forum of 2014. As my project develops
further, I plan on presenting at regional or national conferences such as the National Conference
of Undergraduate Research or the American Society of Cell Biology Annual meeting. In addition
to conference presentations, I will be completing a research thesis for the Honors fellows
program and aim to work towards a peer-reviewed publication.
Part III: Feasibility
Feasibility statement:
This project is very feasible, because of the resources and connections that I have as well
as my motivation and love for this project. When interviewing to be a part of the Periclean
Scholars class of 2015, I told our mentor Dr. Warner that I had a passion for understanding
poverty- what systems are behind it existence and what issues it creates. I also told him that I felt
that because I had that passion, it was my responsibility to do something about it. By doing this
project I am gaining an understanding of an effect and a perpetuating cause of poverty. At the
same time, I also have a fascination for the chemical interactions within the body. This project is
a combination of both of my passions, which will be a strong motivator throughout the entire
project. I am also confident in my ability to complete the experiments necessary to gaining an
understanding of aflatoxins’ effects. I have already begun working in the lab with Dr. Train,
learning various cell culture and maintenance techniques of which I can build upon as we move
forward which more challenging assays. I have been able to learn techniques from both the
seniors in my lab and from Dr. Train, which prepare me for the experiments that I will run for
this project.
I am also confident in the feasibility of this project because of the resources available to
me. The Biology Department at Elon is already equipped with equipment and materials that I
need, such as the microcentrifuge, the -80oC freezer, blotting kits, cell culturing plates,
micropipettors, and some basic antibodies. These have allowed me to start working in the lab on
my project and will allow me to do more investigations in the future. Through collaboration with
other departments, I will also be able to access more equipment, materials, and knowledge that
will aid in the success of this project. By communicating with the Chemistry department, I would
be able to use equipment such as the flow cytometer, which would allow me to quickly analyze
the growth and activity of the cells that I am studying. In the Environmental Science department,
Professor Steve Moore has done research at N.C. State on the effects of aflatoxin on animals; his
research may help me gain further insight into my project. Within the lab, Dr. Train has
extensive background involving the identification of apoptotic cells and the effects of protective
factors on immune cells both of which are major components of this project.
Budget:

Cells
o Jurkat E6.1 Cell Line human
o Human Peripheral Blood Mononuclear Cells
o Other Cell Lines
Total

Western Blot Supplies
o TGX precast gels
o Tris-Glycine-SDS Buffer
o Phosphate buffered saline
Total

Antibodieso Phospho- p44/42 MAPK (Erk ½)
o Caspases
o Apoptosis Antibody Sampler Kit
$369.50
$435.00
$350.00
$1154.50
$208.20
$83.80
$108.00
$400.00
$301.00
$500.00
$489.00
Total

Cell Culturing Supplies
o Tissue Culture Dishes
o 12-well cell culture plates
o Fetal Bovine Serum (500 mL)
o RPMI media
Total

Chemical Supplies
o Aflatoxin
o Other
Total

Equipment
o Micropipetters
o Other Equipment
Total

Conferences
o ASCB conference submission
o NCUR Conference submission
o Posters
Total

Travel
o Flight from Raleigh to Philadelphia Round Trip
o Round trip flight to NCUR 2014
o Hotel Expenses
o Food Expenses
Total

$1290.00
$250.90
$296.60
$304.00
$104.00
$955.50
$100.00
$300.00
$400.00
$800.00
$500.00
$1300.00
$100.00
$100.00
$100.00
$300.00
$300.00
$400.00
$300.00
$200.00
$1200.00
Tuition
o Fall 2013-Spring 2014
o Fall 2014-Spring 2014
$4000.00
$4000.00
Total
TOTAL
$8000.00
$15,000.00
Timeline:
Summer 2013
Fall 2013
Winter 2014
Spring 2014
Summer 2014
Fall 2014
Winter 2015
Spring 2015
Proposed Experiences
Summer Undergraduate
Research Experience
Preliminary studies to determine
the effect of aflatoxin on Jurkat T
cell growth and viability
HNR498- 2 credits
Potentially Attend the American
Society for Cell Biology Annual
Meeting in New Orleans, LA
Expand studies on Jurkat T cells
to determine intracellular
signaling events.
Public Health Practicum:
Community Rural Health Project,
India
HNR 298- 2 credits
Continue studies on growth,
viability and signaling. Begin
preliminary work on primary
immune cells.
Attend NCUR Conference at
University of Kentucky
Participate in an REU at a
Research Institution
HNR 298- 2 credits
Attend and Potentially Present at
the ASCB Annual Meeting in
Philadelphia, PA. Dec. 6-10, 2014
Continue work on primary
immune cells.
Continue work on project while
taking a winter term course
HNR 298- 2 credits
Perform necessary replications,
complete studies in the laboratory
and write up results.
Proposed Product(s)
SURE Presentations
Submission of Abstract to the
National Conference of
Undergraduate Research (NCUR)
Student Undergraduate Research
Forum presentation
Submission of abstract to American
Society of Cell Biology (ASCB)
Potential Conference Presentation
at ASCB Annual Meeting
Synthesis of research into an
Honors Thesis
Submission of publication to PeerReviewed journal
List of Sources:
1
Jiang, Y, Jolly, P, Ellis, W, Wang, J, Phillips, T, Williams, J. (2004). Aflatoxin B1 albumin
adduct levels and cellular immune status in Ghanians. International Immunology. 17(6):807814.
2
Williams, J, Phillips, T, Jolly, P. Stiles, J. Jolly, C, Aggarwal, D. (2004). Human aflatoxicosis
in developing countries: a review of toxicology, exposure, potenital health consequences, and
interventions. The American Journal of Clinical Nutrition. 80:1106-22.
3
Bruneau, J, Stack, E, )’Kennedy, R, Loscher, C. (2012) Aflatoxins B1, B2, and G1 modulate
cytokine secretion and cell surface marker expression in J774A.1 murine macrophages.
Toxicology in Vitro. 26:686-693.
4
Turner P, Moore S, Hall A, Prentice A, Wild C. (2003).Modification of Immune Function
through Exposure to Dietary Aflatoxin in Gambian Children. Environmental Health
Perspectives. 111(2):217-220.
5
Jiang Y, Jolly P, Preko P, Wang J, Ellis W, Phillips T, Williams J. (2008).Aflatoxin-Related
Immune Dysfunction in Health and in Human Immunodeficiency Virus Disease. Clinical and
Developmental Immunology 2008: doi: 10.1155/2008/790309.
6
Cusumano V, Rossano F, Merendino RA, Arena A, Costa GB, Mancuso G, Baroni A, Losi E.
(1996).Immunobiological activities of mould products: functional impairment of human
monocytes exposed to aflatoxin B1. Research in Microbiology. 147:385-391.
7
Han SH, Jeon YJ, Yea SS, Yang KH. (1999).Suppression of the interleukin-2 gene expression
by aflatoxin B1 is mediated through the down regulation of the NF-AT and AP-1 transcription
factors. Toxicology Letters. 108:1-10.
8
Gross-Steinmeyer K, Eaton D. (2012). Dietary modulation of the biotransformation and
genotoxicity of aflatoxin B1. Toxicology. 299: 69-79.
9
Liu Y, Wu F. (2010). Global Burden of Aflatoxin-Induced Hepatocellular Carcinoma: A Risk
Assessment. Environmental Health Prospectives. 118: 818-824.
10
Hatori Y, Sharma R, Warren RP. (1991). Resistance of C57B1/6 mice to immunosuppressive
effects of aflatoxin B1 and relationship with neuroendocrine systems. Immunopharmacology.
22: 127-136.
11
Johnson NM et. al. (2010). Aflatoxin and PAH exposure biomarkers in a U.S. population with
a high incidence of hepatocellular carcinoma. Science of the Total Environment. 408: 60276031.