Arsenic and Apoptosis in PHLC-1 cells

1
Arsenic and Apoptosis in PHLC-1 cells Yeong-Nam
Jeong and Dr. Elizabeth Murray Marshall
University, Integrated Science and Technology
Department
Abstract Background Arsenic has been well
characterized as a carcinogen in eukaryotic
cells. Many researches propose that arsenic
caused tumor or various cancer types using
mammalian cells. We are studying using the PHLC-1
(Poeciliopsis lucida hepatocellular carcinoma)
cell lines which are derived from a liver tumor
from Poeciliopsis lucida because fish can be
affected very directly by environmental
contamination in water. The previous study, we
detected DNA damage from comet assay. The cells
were exposed in low levels of arsenic (5uM and
10uM of As2O3 and 25uM and 50uM of As2O5) for one
and two hours and compared with 1X PBS controls.
Lau et al. reported that arsenic trioxide caused
apoptosis in rat lung epithelial cells. Their
results suggest low arsenic levels may cause
cancer and high arsenic may cure cancer, because
of induction of apoptosis. Methods Confocal
Assay PHLC-1 cells were grown in WillCo glass
bottom dishes. Cells were exposed to 5uM As2O3
for varying lengths of time (1 to 6 hr). Cells
were treated with the Calbiochem
Fluorescein-FragELTM DNA Fragmentation Detection
Kit. It is a fluorescence system for labeling of
DNA breaks in apoptotic nuclei in cell
preparations directly fixed in WillCo glass
bottom dishes. We detected and analyzed the
apoptotic cells using the Bio-Rad Confocal
Microscope. Agarose gel DNA fragmentation assay
PHLC-1 cells exposed to 5uM As2O3 for varying
lengths of time (1 to 6 hr) were lysed, and the
DNA was purified by phenolchloroform extraction
followed by ethanol precipitation. In 1.0
TBEagarose gel, the DNA was electrophoresed and
the gel was stained with ethidium bromide and
imaged using the Bio-Rad GelDoc System.
Results Apoptosis is a highly regulated pathway
that maintains cell proliferation in balance with
cell death. If apoptosis is stimulated or
suppressed inappropriately, cancer or abnormal
development could result. Arsenic induces
apoptosis in mammals. This metal is a commonly
found in waters in West Virginia, Ohio and
Kentucky that are inhabited by fishes. Although
apoptosis occurs in fishes exposed to metals, the
specific effects of this element on the apoptotic
cascade is unknown. PHLC-1 cells are fish cells
used to study toxicology. This research will
report the use of these cells to monitor
apoptosis induced by As2O3. Significance Our
laboratory will use these research results on the
effects of arsenic on the fish apoptosis, to
develop speedy, cost-effective cytotoxicity tests
to examine apoptosis in fishes inhabiting
polluted streams in Appalachia.

• Methods
• Cell culture
• P. lucida cell lines were obtained from ATCC and
  cultured in humidified CO2 incubator at 30 C.
  Cells were grown in Minimal Essential Medium
  Eagle with 1mM Sodium pyruvate, 2 mM L-glutamine
  and 1500 mg sodium bicarbonate/L supplemented
  with 5 Fetal Calf serum and 1 Pen Strep. Cells
  were split every 2-3 days. Experiments were
  performed on confluent cells.
• Cell Growth Assay
• Cells were seeded at 12 well. 1x105 cells/ml and
  3x104 cells/ml were seeded in each of 6 wells.
  This assay was duplicated. For counting the
  cells, these cells were trypsinized. A standard
  Hemocytometer Cell Count Calculator was used to
  count cells at intervals after initial plating.
  These cells were stained with trypan blue to
  determine cell viability. Initial concentrations
  of cells plated were varied to determine optimal
  growth rate.
• Apoptosis Kit
• For apoptosis assay we used the FragELTM DNA
  fragmentation Detection Kit, Fluorescent TdT
  Enzyme (QIA 39) from CalBiochem. There are four
  steps and they are
• Fixation 2. Permeabilization 3. Enzymatic
  labeling reaction 4. Termination
• First, cells were fixed with 4 formaldehyde in
  PBS. To allow the enzyme and substrates to enter
  the cell, we permeabilized them by Proteinase K
  treatment 20ug/ml in 10mM pH 8.0 Tris buffer for
  5 minutes. Enzymatic addition of labeled
  nucleotides was carried out by using Terminal
  Deoxynucleotidyl Transferase (TdT) and
  fluorescein labeled deoxynucleotides.
  Equilibration of cells was done in the 1X TdT
  (Terminal Deoxynucleotidyl Transferase) buffer
  for 10-30 minutes at room temperature. After
  equilibration, enzyme and nucleotides were
• added (60ul TdT Labeling Reaction) the mixture
  was incubated at 37oC for 1-1.5 hours. The fixed
  and labeled cells were washed in 1x TBS 3 times
  for 1 minute and mounted with a glass cover slip
  using Mounting Media.
• Confocal Microscopy
• For this assay, the Bio-Rad MRC1024 Confocal
  Scanning Microscope was used. Since we used
  fluorescein as our fluorescent label (ex. max
  490nm, em. Max 525nm), we set up our microscope
  with the 488nm excitation line and the 522/329nm
  band pass emission line. The image was gotten on
  40X with oil. We use Carl Zeiss AIM software and
  Image J for making images better.

Introduction Arsenic is a well-characterized as a
carcinogen in mammalian cells. Arsenic is
soluble in water and is a colorless, flavorless,
and unscented solution. The U.S. EPA had
established the maximum contaminant level (MCL)
for arsenic in drinking water of 50 ppb (50 ug/L)
which changed to 10 ppb (10 ug/L) in January 2006
8. Arsenic has been linked to several forms of
cancer (bladder, lungs, skin, kidney, nasal
passages, liver, and prostate). Arsenic exposure
is associated with cardiovascular, pulmonary,
immunological, and neurological, and endocrine
problems. Inorganic arsenic has both acute
(short-term) and chronic (long-term) toxicity
8. How arsenic causes cancer is not well
understood. We chose the PLHC-1 cell lines
because fish are informative for environmental
carcinogenesis research, since fish populations
live in polluted habitats 7. Liver cells are
also useful for toxicity assays. Jia et al.
reports that the two most common forms of
inorganic arsenic forms found in drinking water,
As2O3 and As2O5, cause DNA damage to rat
astroglia cells (primary nerve cells) as detected
by the Comet Assay 9. We have already detected
DNA damage from Comet Assay using PLHC-1 cells,
but we could not determine if this was from
necrosis or apoptosis. So, we decided to do this
experiment using the Bio-Rad MRC 1024 Confocal
Scanning Microscope to figure out if Arsenic
causes apoptosis in fish. Apoptosis is a highly
regulated pathway that maintains cell
proliferation in balance with cell death. If
apoptosis is stimulated or suppressed
inappropriately, cancer or abnormal development
could result. The arsenic induced apoptosis in
mammals are well studied. Apoptosis occurs in
living fishes exposed to metals, but the specific
effects of this element on the apoptotic cascade
is not understood well. Cell culture cells are a
useful model for apoptosis since they are easier
to work with than whole fish. This research will
report the use of PLHC-1 cells to monitor
apoptosis induced by As2O3. We detected
apoptosis for treat terminal deoxynucleotidyl-medi
ated transferase (TdT) fluorescent Enzyme
labeling using FragEL DNA fragmentation Detection
Kit from CalBiochem. To obtain apoptosis images,
we used the Bio-Rad MRC1024 Confocal Scanning
Microscope.
Fig 8. A. No arsenic treatment control cells.
B. 5um As2O3 1hr. C. 5um As2O3 3hr Occasional
apoptotic cells are seen in the untreated cells.
On the 1 hour treatment cells, note blobbing and
shrinking apoptotic cells. Many apoptotic cells
are visible. On the 3 hours treatment cells,
these cells look like they are almost dead from
apoptosis.
DNase Fragmentation Assay did not show
significant DNA degradation in apoptotic ladders.
Liu et al. suggested that DNA fragmentation
assays were for 48 or 72 hours, so we plan to
repeat these experiments 6.
Results
Discussion There have not been many studies of
apoptosis in tissue cultured fish cells.
Recently there have been two papers published on
this topic. Liu et al. discusses using grass
carp cell lines in environmental toxicology using
atrazine, an herbicide which pollutes water 6.
They found that there was a dose response to
increased amounts of atrazine. Embry et al. shows
that apoptosis mechanism in fish cells may be
very different that in mammalian cell lines. P53
expression in apoptosis is not induced by
chemotherapy agents in PLHC-1 cell and in primary
liver cells from rainbow trout. Arsenic induced
apoptosis is reported to be p53 independent in
mammalian cell lines 5. Arsenic induces AP-I
activations through activation of MAP (Mitogen
Activated Protein) kinases and PKC (protein
kinase C) 7. This previous research shows that
arsenic is a carcinogen, but also induces
apoptosis in tumor cells. We are interested in
continuing this research. We plan a cytotoxicity
assay, arsenic induced-apoptosis assay using
As2O3 and As2O5, repeat our DNA fragmentation
assay for longer exposure, an arsenic exposure
cell growth assay, and a Western blot for
detecting p53 in PLHC-1 cell lines exposed to
arsenic.
Fig 5. Normal appearance of cultured PHLC-1 cells
from liver tumor of the topminnow. Note, the
morphology seen here is somewhat different than
that seen in our experimental groups that were
grown to confluence.
References 1. www.nativefish.org/Gallery 2. X.
Chris Le, Xiufen Lu, Xing-Fang Li. Arsenic
Spectation. Americal Chemical Society. 2004
26A-33A 3. Andy T.Y. Lau, Muyai Li, Ronglin Xie,
Quing-Yu He, Jen-Fu Chiu. Opposed
arsenic-induced signaling pathways promote cell
proliferation or apoptosis in cultured lung
cells. Carcinogenesis. 2004 25(1)21-28 4. Susan
M. Dibartolomeis, James P. Mone. Apoptosis A
Four-Week Laboratory Investigation for Advanced
Molecular and Cellular Biology Students. Cell
Biology Education. 2003 2 275-295 5. Zigang D.
The Molecular Mechanisms of Arsenic-Induced Cell
Transformation and Apoptosis. Environmental
Health Perspectives. 2002 110(5) 757-759 6.
Xin-Mei Liu, Jian-Zhong Shao, Li-Xin Xian,
Xian-Yong Chen. Cytotoxic Effect and Apoptosis
Induction of Atrazine in a Grass Carp
(Ctenopharyngodon idellus) Cell Line. WILEY
InterScience. 2006. 80-89 7. M Rau Embry, SM
Billiard, RT Di Giulio. Lack of p53 induction in
fish cells by model chemotherapeutics. Oncogene.
2006 25 2004-2010 8. http//www.epa.gov/watersec
urity/guide/chemicalsensorforarsenic.html 9. Jin
Y, Sun G, Li X, Li G, Lu C, Qu L. Study on the
toxic effects induced by different arsenicals in
primary cultured rat astroglia. Toxicol Appl
Pharmacol. 2004196396-403.
Fig 6. These are representative cell growth
assay plots for PHLC-1 cells. The different plot
lines are a measure of growth rate resulting from
variation in the initial concentration of cells
plated in medium. We plan to determine the
difference in Cell Growth using arsenic exposed
cells with standard 96 well cytotoxicity assay.
Fig 2. High magnification image of PHLC-1 cell
nucleas showing typical apoptotic bodies composed
of nuclear chromatin. Here, as in Fig 7 8, the
green fluorescence indicates enzymatic labeling
of fragmented DNA (scale bar is related to grey
image, fluorescent inset is slightly enlarged).
Fig 1. Arsenic-induced signal transduction
pathways and their role in cell transformation
and apoptosis 5.
A.
B.
Acknowledgements Thanks to David Neff and Dr.
Norton for assisting with use of the Bio-Rad MRC
1024 Confocal Scanning Microscope to figure out
the apoptosis assay.
Fig 7. Apoptotic endonucleases have a
fragmenting effect on the cellular DNA by
producing the typical DNA ladder and also
generate free 3-OH groups at the ends of DNA
fragments. These 3-OH ends are the target for
our labeling reaction. A. These HL-60 cells were
prepared as a positive control for apoptosis as
indicated by the TdT assay (cells were provided
in assay kit). B. These PHLC-1 cells are
untreated but show a low rate of apoptosis that
normally occurs cell cultures. Notice the
clearly defined blebs of nuclear material.
Fig 3. Poeciliopsis lucida, Desert topminnow 1.
Fig 4. Comet Assay. This assay represents the DNA
damages.
This is a project for CHM 583 class.